[Pharmacokinetic and pharmacodynamic synergism between neuropeptides and lithium in the neurotrophic and neuroprotective action of cerebrolysin].

OBJECTIVE: To study the synergism between neuropeptides and lithium ions. MATERIAL AND METHODS: An experimental model of stroke (chronic bilateral occlusion of the common carotid arteries in rats), neuronal culture studies, histomorphological analyses, determination of micronutrient profile of brain substrates were used. RESULTS: A complex of experimental studies revealed that the effect of cerebrolysin is influenced by the synergism between lithium ions and the neuropeptide contentof this drug. Pharmacokinetic synergism promotes the accumulation of lithium in brain tissues during cerebrolysin treatment. The existence of the pharmacokinetic synergism is evident from the potentiation of neuroprotective effects of the drug under the action of lithium ions established in the model of stroke. CONCLUSION: Lithium ions potentiate neuroprotective effects of cerebrolysin.

Blood-brain transfer and antinociception of linear and cyclic N-methyl-guanidine and thiourea-enkephalins.

Enkephalins are active in regulation of nociception in the body and are key in development of new synthetic peptide analogs that target centrally located opioid receptors. In this study, we investigated the in vivo blood-brain barrier (BBB) penetration behavior and antinociceptive activity of two cyclic enkephalin analogs with a thiourea (CycS) or a N-methyl-guanidine bridge (CycNMe), and their linear counterparts (LinS and LinNMe) in mice, as well as their in vitro metabolic stability. (125)I-LinS had the highest blood-brain clearance (K1=3.46µL/gmin), followed by (125)I-LinNMe, (125)I-CycNMe, and (125)I-CycS (K1=1.64, 0.31, and 0.11µL/gmin, respectively). Also, these peptides had a high metabolic stability (t1/2>1h) in mouse serum and brain homogenate, and half-inhibition constant (Ki) values in the nanomolar range with predominantly µ-opioid receptor selectivity. The positively charged NMe-enkephalins showed a higher antinociceptive activity (LinNMe: 298% and CycNMe: 205%), expressed as molar-dose normalized area under the curve (AUC) relative to morphine, than the neutral S-enkephalins (CycS: 122% and LinS: 130%).

A prodrug nanoparticle approach for the oral delivery of a hydrophilic peptide, leucine(5)-enkephalin, to the brain.

The oral use of neuropeptides to treat brain disease is currently not possible because of a combination of poor oral absorption, short plasma half-lives and the blood-brain barrier. Here we demonstrate a strategy for neuropeptide brain delivery via the (a) oral and (b) intravenous routes. The strategy is exemplified by a palmitic ester prodrug of the model drug leucine(5)-enkephalin, encapsulated within chitosan amphiphile nanoparticles. Via the oral route the nanoparticle-prodrug formulation increased the brain drug levels by 67% and significantly increased leucine(5)-enkephalin's antinociceptive activity. The nanoparticles facilitate oral absorption and the prodrug prevents plasma degradation, enabling brain delivery. Via the intravenous route, the nanoparticle-prodrug increases the peptide brain levels by 50% and confers antinociceptive activity on leucine(5)-enkephalin. The nanoparticle-prodrug enables brain delivery by stabilizing the peptide in the plasma although the chitosan amphiphile particles are not transported across the blood-brain barrier per se, and are excreted in the urine.

Delivery of neuropeptides from the periphery to the brain: studies with enkephalin.

Many peptides with the potential of therapeutic action for brain disorders are not in clinical use because they are unable to cross the blood-brain barrier (BBB) following peripheral administration. We have developed two potential strategies for the delivery of peptides to the brain and demonstrated their feasibility with enkephalins. In the first approach, designated induced reversible lipophilization, Leu/Met Enkephalins were converted to 9-fluorenylmethoxycarbonyl (Fmoc) derived lipophilic prodrug analogues, which undergo slow, spontaneous hydrolysis under physiological conditions, generating the native agonists. In contrast to Enkephalin, Fmoc-Met-Enkephalin was found to facilitate an analgesic effect following intraperitoneal administration in mice. Fmoc-Leu-Enkephalin was not analgesic. In the second approach, Enkephalin was linked to BBB transport vectors through an Fmoc based linker spacer, forming conjugates that slowly release Enkephalin under physiological conditions. A pronounced antinociceptive response was thus obtained following intraperitoneal administration of either cationized-human serum albumin-Fmoc-Enkephalin or polyethylene glycol(5)-Fmoc-Enkephalin. Derivatives of Enkephalin covalently linked to the same BBB-transport vectors through a stable (nonreversible) chemical bond were not analgesic. In summary, we have demonstrated that lipophilicity can be conferred to hydrophilic peptides to a degree permitting the permeation of the BBB by passive diffusion, without the drawback of agonist inactivation, which is often caused by irreversible derivatization. Similarly, in the second strategy, the conjugation to BBB-permeable vectors overcomes the obstacle of peptide inactivation by releasing the active form in the central nervous system.

Intrathecal antinociceptive interaction between the NMDA antagonist ketamine and the opioids, morphine and biphalin.

Biphalin is an opioid peptide analogue that currently is under clinical development as a new type of site-directed analgesic. In rats, the intrathecal (i.t.) analgesic potency of biphalin is 1000-fold greater than morphine. Such a high activity may reflect this compound's activation of three types of opioid receptors (mu, delta and kappa). NMDA receptors also play an important role in nociceptive processing. Therefore, we investigated in rats whether an NMDA antagonist may influence biphalin-induced antinociception. In the present study, ketamine was chosen because of the widespread safe use of this drug in clinical practice. I.t. application of ketamine alone had relatively little analgesic effect and its excitatory effects limited possible doses of the drug. Co-administration of ketamine with biphalin or morphine produced markedly greater antinociception than biphalin or morphine alone in acute, thermal tail flick testing. These results suggest that NMDA antagonists may be useful potentiators of biphalin analgesia. Thus, to obtain the same spinal antinociceptive effect, lower doses of biphalin or morphine are required when ketamine is co-administered.

High airway-to-blood transport of an opioid tetrapeptide in the isolated rat lung after aerosol delivery.

The airway-to-blood absorption of the mu-selective opioid tetrapeptide agonist Tyr-D-Arg-Phe-Phe-NH(2) (MW 631) was investigated in the isolated, perfused, and ventilated rat lung model. The lung metabolism of the peptide was compared after airway and vascular delivery. The concentrations of the parent tetrapeptide and five of its metabolites in the perfusate and in bronchoalveolar lavage fluid were analyzed by LC-MS. The metabolism of the peptide was higher after delivery to the airways compared to vascular delivery. However, the tetrapeptide was highly transported from the air-to-blood side to an extent of 47.8 +/- 10.7% in 2 h. In conclusion, the results prompt investigations of the pulmonary route as a successful alternative to parenteral delivery for this tetrapeptide.

Regional differences in bioavailability of an opioid tetrapeptide in vivo in rats after administration to the respiratory tract.

TArPP (Tyr-D-Arg-Phe-Phe-NH(2)), 1-10 micromol/kg, was administered to anesthetized rats by nasal microinfusion, intratracheal microinfusion, intratracheal nebulization, aerosol inhalation, and i.v. bolus and infusion. Plasma concentrations of TArPP and its deamidated metabolite were determined by LC-MS-MS. Regional differences in bioavailability (F), first-pass metabolism, and absorption rate were found for TArPP after delivery to the respiratory tract. Absorption was rapid after both pulmonary and nasal administration (t(max) approximately 10-20 min). After nasal microinfusion, F was 52 +/- 9%. For all the pulmonary groups, F was higher (72-114%). First-pass metabolism of TArPP was lower in the lung than in the nasal cavity. It is evident that the pulmonary route is attractive for successful systemic delivery of small, hydrophilic and enzymatic susceptible peptides.

Combined effect of oleic acid and polyethylene glycol 200 on buccal permeation of [D-ala2, D-leu5]enkephalin from a cubic phase of glyceryl monooleate.

The combined use of the lipophilic permeation enhancer, oleic acid together with polyethylene glycol 200 (PEG 200) as a co-enhancer and incorporated into the cubic liquid crystalline phase of glyceryl monooleate was investigated in the ex vivo buccal permeation of [D-Ala2, D-Leu5]enkephalin (DADLE) through porcine buccal mucosa mounted in a Franz cell. The addition of oleic acid (1%) and PEG 200 (1-10%) did not change the intact appearance of the cubic phase. PEG 200 increased the aqueous solubility of oleic acid incorporated into the cubic phase and hence promoted the transport of oleic acid into the porcine buccal mucosa. The solubilising effect of PEG 200 on oleic acid incorporated into the cubic phase was dependent on the PEG 200 concentration but was non-linear. The buccal permeation flux of DADLE significantly increased when 5% (P<0.01) or 10% (P<0.001) of PEG 200 was co-administered with 1% oleic acid compared with the cubic phase containing 1% oleic acid alone. The present results suggest that PEG 200 enhances the action of the lipophilic permeation enhancer oleic acid and that the combination of oleic acid and PEG 200 as a co-enhancer can be a useful tool to improve the membrane permeability in the buccal delivery of peptide drugs using a cubic liquid crystalline phase of glyceryl monooleate and water.

The effect of halogenation on blood-brain barrier permeability of a novel peptide drug.

The utility of a drug depends on its ability to reach appropriate receptors at the target tissue and remain metabolically stable to produce the desired effect. To improve central nervous system entry of the opioid analgesic [D-Pen2, L-Pen5, Phe6] Enkephalin (DPLPE-Phe), our research group synthesized analogs that had chloro, bromo, fluoro, and iodo halogens on the para positions of the phenylalanine-4 residue. This study reports on investigation of the effect of halogenation on stability, lipophilicity, and in vitro blood-brain barrier permeability of a novel enkephalin analog DPLPE-Phe. The stability of each halogenated DPLPE-Phe analog as well as the amidated and nonamidated parent peptide was tested in plasma and brain. All peptides tested had a half-time disappearance >300 min except for DPLPE-Phe-NH2, which was found to have a half-life of 30 min in plasma. Octanol/saline distribution studies indicated addition of halogens to DPLPE-Phe-OH significantly increased lipophilicity except for p-[F-Phe4]DPLPE-Phe-OH. p-[Cl-Phe4]DPLPE-Phe-OH exhibited the most pronounced increase in lipophilicity. Para-bromo and para-chloro halogen additions significantly enhanced in vitro blood-brain barrier permeability, providing evidence for improved delivery to the central nervous system.

Topical opioids in mice: analgesia and reversal of tolerance by a topical N-methyl-D-aspartate antagonist.

In addition to its central actions, morphine has important peripheral effects. To examine peripheral analgesic mechanisms, we developed a topical opioid paradigm in which the tail was immersed in a dimethyl sulfoxide (DMSO) solution containing various drugs. Alone, DMSO was inactive in the tail-flick assay in mice. DMSO solutions containing morphine and peptides such as [D-Ala2,MePhe4, Gly(ol)5]enkephalin (DAMGO) produced a potent, dose-dependent analgesia with the radiant heat tail-flick assay. The actions of the drugs were local. Analgesia was observed only in regions of the tail exposed to the solution and not in more proximal unexposed portions of the tail. Immersion of the tail in a solution containing either 125I-labeled morphine or 125I-labeled DAMGO revealed no detectable uptake of radioactivity into the brain, spinal cord, or blood. In the tail, radioactivity was limited only to the regions actually immersed in the solutions. The topical drugs potentiated systemic agents, similar to the previously established synergy between peripheral and central sites of action. Local tolerance was rapidly produced by repeated daily exposure of the tail to morphine. Topical morphine tolerance was effectively blocked by the N-methyl-D-aspartate (NMDA) antagonist MK801 given either systemically or topically but not intrathecally. The ability of a topical NMDA antagonist to block local morphine tolerance suggests that peripheral NMDA receptors mediate topical morphine tolerance. Morphine was cross-tolerant to DAMGO, but not to morphine-6beta-glucuronide, implying different mechanisms of action. These observations are significant in the design and use of opioids clinically.

Enhanced antinociception of the model opioid peptide [D-penicillamine] enkephalin by P-glycoprotein modulation.

PURPOSE: This study was conducted to examine the influence of P-glycoprotein (P-gp) modulation on the pharmacodynamics of the model opioid peptide DPDPE. METHODS: Mice (n = 5-7/group) were pretreated with a single oral dose of the P-gp inhibitor GF120918 (25 or 250 mg/kg) or vehicle. 3H-DPDPE (10 mg/kg) or saline was administered 2.5 hr after pretreatment. Antinociception was determined, and blood and brain tissue were obtained, 10 min after DPDPE administration. RESULTS: A significant difference (p < 0.001) in DPDPE-associated antinociception was observed among mice pretreated with a 25- (83 +/- 16% MPR) or 250- (95 +/- 5% MPR) mg/kg dose of GF120918 in comparison to mice pretreated with vehicle (24 +/- 14% MPR) or mice receiving GF120918 without DPDPE (12 +/- 8% MPR). A significant difference (p < 0.01) in brain tissue DPDPE concentration also was observed among treatment groups [25 +/- 6 ng/g (vehicle), 37 +/- 11 ng/g (25 mg/kg GF120918), 70 +/- 8 ng/g (250 mg/kg GF120918)]. In contrast, blood DPDPE concentrations were not statistically different between groups (678 +/- 66, 677 +/- 130, and 818 +/- 236 ng/ml for vehicle, GF120918 [25 mg/kg], and GF120918 [250 mg/kg], respectively). A single 100-mg/kg i.p. dose of (+)verapamil increased the brain:blood DPDPE concentration ratio by approximately 70% relative to saline-treated control mice (0.139 +/- 0.021 vs. 0.0814 +/- 0.0130, p < 0.01), a change in partitioning similar to that observed with the low dose of GF120918. These data provide further support for a P-gp-based mechanism of brain:blood DPDPE distribution. CONCLUSIONS: The present study demonstrates that GF120918 modulates blood-brain disposition and antinociception of DPDPE. Coadministration of a P-gp inhibitor with DPDPE may improve the pharmacologic activity of this opioid peptide.

Transport of the delta-opioid receptor agonist [D-penicillamine2,5] enkephalin across the blood-brain barrier involves transcytosis1.

The delta opioid receptor antagonist [D-penicillamine2,5]enkephalin (DPDPE) is an enzymatically stable peptide analogue of Met-enkephalin. DPDPE uses a saturable transport mechanism to cross the blood-brain barrier (BBB), though the exact mechanism is not fully understood. The aim of the present study was to identify the mechanism by which DPDPE enters the brain. The effect of phenylarsine oxide (PAO), an endocytosis inhibitor, on the transport of [3H]DPDPE was investigated using both in vitro and in situ transport studies. Two in vitro models of the BBB utilizing primary bovine brain microvascular endothelial cells (BBMEC) were studied. [3H]DPDPE permeability across monolayers of BBMEC grown on polycarbonate filters was studied. PAO significantly reduced the permeability of [3H]DPDPE across the monolayer. PAO also reduced the uptake of [3H]DPDPE into BBMEC cells, without affecting binding to the cells. The in situ perfusion model of the BBB was also studied, PAO reduced DPDPE uptake by the brain in a dose-dependent manner. These studies indicate that DPDPE enters the brain via an energy-dependent transcytotic mechanism.

Effects of beta-funaltrexamine on dose-effect curves for heroin self-administration in rats: comparison with alteration of [3H]DAMGO binding to rat brain sections.

These studies were undertaken to determine the effects of mu-opioid receptor depletion through irreversible alkylation on the dose-effect curve for heroin self-administration. Heroin maintained responding in rats with an inverted U-shaped dose-effect curve and administration of 10 nmol of beta-funaltrexamine i.c.v. (beta-FNA) significantly increased the ED50 on the ascending limb from 1.9 to 5.3 micrograms/infusion, and from 24.3 to 211.8 micrograms/infusion on the descending limb. Administration of saline i.c.v. produced no effect on heroin self-administration. Administration of 40 nmol of beta-FNA increased the ED50S from 5.1 to 33.9 and from 14.4 to 502.8 micrograms/infusion on the ascending and descending portions of heroin's dose-effect curve, respectively. beta-FNA (40 nmol, i.c.v.) had no effect on cocaine self-administration. [3H]DAMGO binding density was decreased in the caudate and nucleus accumbens by 29 or 54% 24 h after administration of 10 or 40 nmol of beta-FNA i.c.v., respectively. The effects of beta-FNA on heroin self-administration were completely overcome by increasing the dose of heroin however, as the shape and slope of the self-administration dose-effect curve was not different when higher doses of heroin were made available for self-administration compared to control data or saline administration. Therefore, there appear to be spare mu-opioid receptors for heroin for the production of its reinforcing effects in rats. Furthermore, the self-administration dose-effect curves returned to control values prior to the return of [3H]DAMGO binding, further suggesting that the full complement of mu-opioid receptors is not necessary for heroin to produce its reinforcing effects. These findings support the existence of spare mu-opioid receptors for heroin in maintaining self-administration in rats.

Altered disposition and antinociception of [D-penicillamine(2,5)] enkephalin in mdr1a-gene-deficient mice.

This study was undertaken to test the hypothesis that P-glycoprotein (P-gp) modulates opioid peptide pharmacodynamics. [D-Penicillamine2, 5]enkephalin (DPDPE) (10 mg/kg i.v.) was administered to mdr1a(-/-) and wild-type mice to assess systemic disposition and antinociception. A subsequent dose-response experiment examined the impact of P-gp on DPDPE antinociception. In addition, the time course of antinociception was determined after a 0.9-mg/kg [mdr1a(-/-) mice] or 24-mg/kg (FVB mice) i.v. dose. Data were fit with a series of pharmacokinetic-pharmacodynamic models to compare the disposition and action of DPDPE in the two mouse strains. A 10-mg/kg dose produced >80% maximum possible response at all time points in mdr1a(-/-) mice; peak antinociception was <20% maximum possible response in FVB mice. DPDPE systemic disposition did not differ between the two mouse strains. Although brain tissue concentrations were 2- to 4-fold higher in mdr1a(-/-) compared to FVB mice, the dose required to elicit comparable antinociception was nearly 30-fold lower in mdr1a(-/-) mice; brain tissue EC50 differed by an order of magnitude in the two mouse strains. Pharmacokinetic-pharmacodynamic modeling indicated that the difference in antinociception between mdr1a(-/-) and FVB mice was a function of DPDPE distribution within brain, as well as between blood and brain, and not due to differences in intrinsic response. The results of this study suggest that DPDPE is a substrate of P-gp, and that P-gp is responsible, in part, for the low penetration of DPDPE into brain. The substantial difference in brain tissue EC50 in the absence vs. presence of P-gp suggests that P-gp modulates DPDPE-associated antinociception at sites other than the blood-brain interface.

Differences between two substrains of AB mice in the opioid system.

Animals from two substrains of AB mice, i.e., ABH/Md and ABG/Md, differ in the occurrence of aggressive behavior. After maturation, male ABH mice regularly exhibited abnormal aggressive behavior making group-housing impossible. In contrast, ABG animals never showed such behavioral patterns. To elucidate the role of opioid mechanisms, we tested the reaction of these animals to morphine in the hot plate test. Moreover, specific DAMGO binding was measured. It was shown that mice from control groups differed significantly in reaction to the thermal stimulus. ABH mice had significantly longer reaction times. With increasing doses of morphine this difference disappeared, suggesting different levels of basal activity in endogenous opioid systems. This is underlined by significantly lower DAMGO binding in aggressive ABH mice. The results suggest that differences in endogenous opioid systems may account for differences in aggressiveness.

Brain and spinal cord distribution of biphalin: correlation with opioid receptor density and mechanism of CNS entry.

Biphalin [(Tyr-D-Ala-Gly-Phe-NH)2] is a bivalent, opioid peptide containing two pharmacophores linked by a hydrazine bridge. When administered intracerebroventricularly, it has been shown to be more potent than morphine and etorphine at eliciting antinociception. Biphalin has also been shown to cross both the blood-brain and blood-cerebrospinal fluid barriers. To understand the basis of biphalin's potency, regional brain and spinal cord distribution studies with [125I-Tyr1]biphalin were performed 5, 20, and 40 min after intravenous bolus injections. A statistically greater amount of [125I-Tyr1]biphalin was detected in the nucleus accumbens compared with other brain regions (p < 0.05). This correlates with the high density of delta- and mu-opioid receptor mRNA and binding sites shown to be expressed in the nucleus accumbens. Also, a statistically greater amount of [125I-Tyr1] biphalin was detected in two other circumventricular organs, the choroid plexus and pituitary, when compared with other brain regions. These studies provide evidence that biphalin can reach not only brain sites, but also spinal sites to elicit antinociception. The overall CNS distribution of [125I-Tyr1]biphalin was decreased with naloxone, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, or naltrindole pretreatment, showing that biphalin detected in the brain and spinal cord is binding to delta- and mu-opioid receptors. Additional in situ brain perfusion experiments identified a saturable component contributing to CNS entry of [125I-Tyr1]biphalin, which could be described by Michaelis-Menten kinetics with a Km of 2.6 +/- 4.8 microM, Vmax of 14.6 +/- 2.89 pmol(-1) x min(-1) x g(-1), and Kd of 0.568 +/- 0.157 microl x min(-1) x g(-1). Brain entry of [125I-Tyr1]biphalin was sensitive to 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and L-phenylalanine, suggesting use of the large neutral amino acid carrier. This work provides evidence that biphalin is a promising, potent analgesic that has a unique mechanism for reaching both spinal and supraspinal opioid receptor sites.

The entry of [D-penicillamine2,5]enkephalin into the central nervous system: saturation kinetics and specificity.

The delta opioid receptor-selective, enzymatically stable peptide [D-Penicillamine2,5]enkephalin (DPDPE) has recently acquired special significance with the identification of a saturable uptake system for this analgesic into the CNS. The aim of the present study was to characterize further the entry of [3H]DPDPE into the brain and CSF by means of a bilateral in situ brain perfusion method. Initial experiments revealed a saturable [3H]DPDPE uptake into the brain that followed Michaelis-Menten type kinetics with a K(m) value of 45.5 +/- 27.6 microM, a V(max) value of 51.1 +/- 13.2 pmol x min(-1) x g(-1) and a K(d) value of 0.6 +/- 0.3 microl x min(-1) x g(-1). Uptake of [3H]DPDPE into the CSF could not be inhibited (K(d) = 0.9 +/- 0.1 microl x min(-1) x g(-1)). Entry of [3H]DPDPE into the CNS was not inhibited in the presence of 10 mM 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid (BCH) or 50 microM ICI 174,864, which suggests that the saturable mechanism does not involve the large neutral amino acid transporter or binding to opioid receptors. It would also appear that [3H]DPDPE is not in competition with either poly-L-lysine or insulin to enter the CNS. However, both of these substances significantly increased the CNS entry of [3H]DPDPE but not that of the vascular space marker [14C]sucrose, and this may have valuable clinical implications. It is not known at present which saturable uptake mechanism is responsible for the CNS entry of [3H]DPDPE, but overall the results suggest a carrier-mediated transport system.

In vitro skin penetration and degradation of enkephalin, elcatonin and insulin.

The work described in this paper was designed to evaluate the relevance of in vitro skin penetration studies of peptides across rat skin. The apparent penetration of three peptides, enkephalin, elcatonin and insulin, in the presence of enhancers was not seen in the in vitro method using Franz diffusion cells. However, when a protease inhibitor was mixed in the receptor fluid, the penetration of enkephalin and insulin was observed. Although insulin penetrated in the presence of enhancers, the penetration was extremely small in quantity and the cumulative amount did not increase with time. When the degradation of peptides in the receptor fluid of Franz cell was estimated, these peptides, especially enkephalin and insulin, were rapidly hydrolyzed and were almost completely lost within 3 h in the absence of an inhibitor, while elcatonin was slowly degraded. The addition of protease inhibitors, such as gabexate (20 mM), camostat (20 mM) or bile salt (taurocholate and deoxycholate, 10 mM), to the receptor fluid inhibited the degradation to a considerable extent, with the first-order rate constants decreased to one-tenth compared with the constants without inhibitors. From the inhibitory study using specific inhibitors, it was clarified that enkephalin and elcatonin were mainly hydrolyzed by aminopeptidases, endopeptidases and serine proteases in the viable skin. Consequently, the results obtained from the in vitro penetration studies without inhibitors did not reflect reliable penetration data. Thus, effective protease inhibitor(s) should be used to obtain the data corresponding to the in vivo transdermal experiment. This methodology will provide a means to eliminate the confounding effect of metabolism in permeation experiments.

Blood-brain disposition and antinociceptive effects of -D-penicillamine2,5-enkephalin in the mouse.

Although intravenous administration of [D-penicillamine2, 5]-enkephalin (DPDPE) produces significant antinociception in rodents, the duration of antinociception is short ( approximately 15 min). The present study was conducted to test the hypothesis that duration of antinociception for DPDPE is determined by both systemic and regional disposition (i.e., blood-brain translocation), and that the magnitude of antinociception is related more closely to concentrations in brain tissue than in blood. Systemic disposition was examined after i.v. administration of DPDPE (10-100 mg/kg) to male CD-1 mice. The relationship between antinociception and concentration in blood and brain tissue was assessed by determining antinociception 10 min after administration of DPDPE (10-100 mg/kg); effect versus brain tissue concentration data were fit with pharmacodynamic models to recover EC50 estimates. In addition, the time course of antinociception, as well as blood and brain tissue concentrations, were examined after an i.v. bolus dose (40 mg/kg) of DPDPE. The systemic disposition of DPDPE was nonlinear; both clearance and volume of distribution were dose-dependent. Antinociception increased proportionately with increasing concentrations of DPDPE in blood or brain tissue, with an EC50 of 1.42 +/- 0.06 microg/g expressed as brain tissue concentration. However, the brain-to-blood concentration ratio also increased with increasing dose, suggestive of saturable translocation of DPDPE across the blood-brain barrier. Antinociception appeared rapidly (within 5 min) and dissipated within approximately 15 min after a 40 mg/kg i.v. dose. These results suggest that rapid elimination from blood and active efflux from brain limit the duration of action of DPDPE.

Lipid membrane permeability of modified c[D-Pen2, D-Pen5]enkephalin peptides.

Permeability coefficients of a series of analogues of a potent opioid peptide, c[D-Pen2, D-Pen5]enkephalin, were measured in a model membrane system. The analogues included hydrophobic amino acid substitutions on position 3. Liposomes of a mixed composition consisting of zwitterionic lipids and cholesterol served as the model membranes. The obtained permeability coefficients range between 0.38 x 10(-12) and 2.9 x 10(-12) cm/s. These data were correlated with the hydrophobicity scale of Nozaki and Tanford (J. Biol. Chem. 246, 1971, 2211-2217) (correlation coefficient = 0.9933) and with determinations of lipid order perturbation by differential scanning calorimetry (correlation coefficient = -0.9779). The reasonably good correlation obtained within the family of analogues substituted on position 3 (Gly, Ala, Leu, Phe) indicates that changes in permeabilities are primarily related to increases in the partition coefficient of the peptide. However, Phe residue added on the N-terminal end of the peptide (position 0) does not appear to follow the observed trend, showing stronger lipid perturbation and lower permeability compared to the Phe3 analog. This observation demonstrates that each class of peptide modifications requires a new basis of permeability analysis and predictions.