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

Abstract

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.

Keywords: Neuropeptide; blood−brain barrier; enkephalin; prodrug; reversible-conjugation; reversible-lypophilization.

Figure 1

Structures and nomenclatures of the Fmoc-Enkephalin analogues investigated in this study. Derivatives are Fmoc-N^α-leucine-Enkephalin (FLE-1077); Fmoc-N^α-methionine-Enkephalin (FME-1091); 3- Thio [11-undecanoic acid], N-3-propiosuccinimido-4-amido-Fmoc-Leu-Enkephalin (C10FME-1095); and S,S′-di-[3-succinimido-N-3-propion-4-amido-Fmoc-Leu-Enkephalin] dithiothreitol (LEFFLED-1031). Dashed arrows indicate the sites of cleavage that take place upon incubation at physiological conditions.

Figure 2

Rate of Enkephalin released from HSA-Fmoc-Enkephalin; PEG₅-Fmoc-Enkephalin; and Fmoc-Met-Enkephalin upon incubation at pH 8.5 and 37 °C. Solutions of HSA-Fmoc-Enkephalin, PEG₅-Fmoc-Enkephalin, or Fmoc-Met-Enkephalin, each containing 100 μg/mL covalently linked Enkephalin, were incubated at 0.1 M sodium-borate buffer at pH 8.5 and 37 °C. At the indicated time points, aliquots (0.1 mL) were analyzed by HPLC for the amount of released Met/Leu-Enkephalin.

Figure 3

Extent and time course of antinociceptive response following intraperitoneal administration of Enkephalins and derivatives. (A) Enkephalins and their Fmoc analogues were administered intraperitoneally (0.2−0.3 mL) to ICR male mice (n = 5 per group), at a dose of 20 μmol/kg body weight, and assayed by the tail flick test. Results are expressed as seconds prior to tail flick. (B) Time course of response after intraperitoneal administration. Each point is the arithmetic mean ± SEM from 5 male ICR mice.

Figure 4

Intraperitoneal administration of Fmoc-derived lipophilic Leu-Enkephalin analogues produces analgesia. (A) The indicated Enkephalins and analogues were administered intraperitoneally, each at a concentration of 10 μmol/kg body weight, and analyzed for their analgesic effect in mice, 40 min after administration. (B) Time course of the antinociceptive response following intraperitoneal administration of HOOC(CH₂)₁₀-Fmoc-Leu-Enkephalin (C10FME-1095). Each group consisted of 5 mice. Each point in the figure is the arithmetic mean ± SEM from 5 male ICR mice.

Figure 5

Analgesic effect of peritoneally administered cationized HSA-Fmoc-Enkephalin. (A) The indicated Leu-Enkephalin analogues, were administered intraperitoneally, each at a concentration of 2 μmol per 1 kg body weight, and analyzed for their analgesic effect 60 min after administration. (B) Time course of the antinociceptive response following intraperitoneal administration of cationized HSA-Fmoc-Leu-Enkephalin. Each point in the Figure is the arithmetic mean ± SEM from 5 male ICR mice.

Figure 6

Reversibly pegylated derivatives of Leu-Enkephalin are analgesic following intraperitoneal administration. The indicated Leu-Enkephalin derivatives were administered at the indicated concentrations, and the antinociceptive response was assayed 60 min after ip administration and compared to that of intraperitoneally administered morphine (10 μmol/kg body weight) and PBS. Each determination is the arithmetic mean ± SEM from 5 male ICR mice.