Peptide Targeting and Delivery across the Blood−Brain Barrier

Peptide Targeting and Delivery across the Blood−Brain Barrier Utilizing ... Publication Date (Web): May 28, 1997 ... Vector-mediated drug delivery t...
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Bioconjugate Chem. 1997, 8, 434−441

434

Peptide Targeting and Delivery across the Blood-Brain Barrier Utilizing Synthetic Triglyceride Esters: Design, Synthesis, and Bioactivity Dinesh Patel,† Brian D. McKinley,† Thomas P. Davis,‡ Frank Porreca,‡ Henry I. Yamamura,‡ and Victor J. Hruby*,† Departments

of

Chemistry

and

Pharmacology,

University

of

Arizona,

Tucson,

Arizona

85721.

Received October 6, 1996X

As an approach to the development of therapeutically useful peptide pharmaceuticals that can penetrate the blood-brain barrier, we have designed and demonstrated the application of a carriertargeting system. We have developed a prodrug design strategy that is designed to utilize membranebound enzymes whereby release of a bioactive peptide from a highly lipophilic triglyceride peptidecarrier is achieved in situ, thus attaining high localized concentrations of the bioactive peptide. Following localization of such a system, normal peptidase and lipase action is utilized to release the active peptide (deltorphin II) intact and in high concentration. At present, the exact mechanisms are unclear, but the observed results in which analgesia is observed following peripheral administration suggest that the active peptide is able to cross the blood-brain barrier and sustain prolonged periods of analgesia as determined by antinociception tests by release of the bioactive peptide. In vitro tests of binding and bioactivity by the peptide conjugate show essentially no potency in either target or control analogues, but potent antinociceptive effects are observed following peripheral administration.

INTRODUCTION

The in vivo site-specific delivery of bioactive analgesic peptides has been a challenging objective for medicinal chemists who strive to utilize the ever-increasing database of highly potent/selective opioid peptide sequences. Ever since the identification of the endogenous enkephalins, numerous successful studies have been reported on the design of highly potent opioid receptor ligands [the reader is referred to a few excellent reviews and the references therein (1-3)]. Very few studies, however, have been reported where attempts have been made to specifically site deliver these peptide analgesics. Those that have been reported have shown varying modest degrees of success (e.g., refs 4-7). A major barrier must be overcome before the promise of these potent and selective ligands can be realized. The blood-brain barrier (BBB) is a unique membrane barrier that tightly segregates the brain interstitial fluid bathing the synapses from the circulating blood. With the exception of the brain and spinal cord, all organs are perfused by capillaries lined with endothelial cells that have small pores for the rapid movement of drugs and solutes into the organ interstitial fluid from the circulation. However, the capillary endothelium of the mammalian brain and spinal cord lack these pores, and therefore drugs can cross the BBB and enter the brain only via two principal mechanisms, either by carriermediated, catalyzed transport or by free diffusion. Peptides generally do not cross the BBB well because these highly polar compounds are not very lipid soluble and often do not have access to specialized BBB receptormediated transport systems. In principle, a peptide neuropharmaceutical that does not cross the BBB may be transported through the BBB by first coupling the active peptide to a BBB transport vector, i.e., a peptide * Author to whom correspondence should be addressed. † Department of Chemistry. ‡ Department of Pharmacology. X Abstract published in Advance ACS Abstracts, May 1, 1997.

S1043-1802(97)00027-X CCC: $14.00

or modified plasma protein that is capable of transversing the BBB via receptor-mediated or absorptive mediated transcytosis through the brain capillary endothelial cell cytoplasm (8-11). The primary aim in any site-specific delivery must be to ensure a selective deposition of the active species at the site of interest. This will in itself ensure a decrease in side effects brought about due to general administration of the drug, and furthermore, this will also decrease the overall dose required since general circulatory loss/ absorption will be lower. Other factors that must be considered in the design of such a species include the prevention of self loss through self-aggregation or micellar formation. In principle, controlled deposition can be achieved by masking or prodrug design, so long as the active molecule remains masked while in circulation, and deposition is accomplished by release of the prodrug enzymatically at or near its site of action. Furthermore, the carrier for such a prodrug complex should have physicochemical properties that make it unique in its carrying capacity, in the sense that when bound to the efficacious component it masks the activity of said component without itself demonstrating undesired side effects. Site-specific delivery of such a prodrug carrier complex is a further complication in the design of such a species. It is perhaps fortunate that endothelial cell membranes are heterogeneous with respect to their content of membrane-bound enzymes distributed around the body (10, 11). Specifically, endopeptidases are localized with respect to their substrate specificity and also in their distribution around blood vessels of the brain. Utilizing the endopeptidase substrate specificity allows for the design of a targeting principal to be incorporated into the prodrug carrier complex. Activation of the complex is accomplished in vivo by removal of a mask by the endothelial membrane-bound enzymes in the locality of interest. Thus, the now-activated carrier complex is available for adsorption into the lipid environment of the cell in this locality. © 1997 American Chemical Society

Peptide Targeting across Blood−Brain Barrier

Peptides, both naturally occurring and designed, often can be highly efficacious and increasingly can be designed to be highly specific in their actions, with few demonstrated undesired side effects. The aim of the present study is to overcome what is probably the one major drawback of using peptides as drugs, and that is their poor ability to cross lipid membranes, specifically the blood-brain barrier. Peptide analgesics that have been found to be super-agonists when administered intracerebro ventricularly (i.c.v.) can show poor activity when administered intraveinously (i.v.). It is generally thought that one of the primary reason for this is their poor ability to cross the BBB even when they are relatively stable to degradation by peptidases. We believe that if the concentration of peptide can be maintained while in circulation, and if the peptide can be delivered into or onto the cell membranes at or near their desired site of action, then there is a higher probability that one would see a greater biological action of the peptide through the simple property of mass action. In this present study, we demonstrate the design, synthesis, and bio-evaluation of a triglyceride peptide carrier prodrug complex that shows greatly improved analgesic activity of a heptapeptide deltorphin over administration of the free peptide. EXPERIMENTAL PROCEDURES

Mouse Vas Deferens (MVD) and Guinea Pig Ileum (GPI) Bioassays. Electrically induced smooth muscle contraction of MVD and strips of GPI longitudinal muscle, myenteric plexus, were used as bioassays (12). In these assays, opioid agonists inhibit the electrically induced smooth muscle contractions in a dose-related manner in both smooth muscle systems. Percent inhibition of electrically induced muscle twitch was calculated as the average contraction height for 1 min preceding the addition of the agonist divided by the contraction height 3 min after exposure to the agonist. IC50 values represent the mean of not less than four tissue samples. IC50 values, relative potency estimates, and their associated standard errors were determined by fitting the mean data to the Hill equation with a nonlinear least-squares method (13). Radioligand Binding. Membranes were prepared from whole brains taken from adult male SpragueDawley rats (250-300 g). All radioligand displacement experiments were run against the 3H-labeled ligands [pClPhe4]DPDPE and CTOP as previously described (14, 15). A minimum of three experiments were conducted for each radioligand. Data obtained from independent measurements were represented as the arithmetic mean ( SEM. Antinociception Studies. Male ICR mice (20-30 g) were used throughout these studies. They were housed in groups of four in Plexiglas boxes, maintained in a lightand temperature-controlled environment, with food and water available ad libitum until the time of antinociceptive testing. The peptide carrier complex and controls were dissolved in a few microliters of methanol and then diluted into physiological saline for peripheral administration via an intraperitoneal (i.p.) route. At the level of methanol used no toxicity is observed. All testing was performed in accordance with the recommendations and policies of the International Association for the Study of Pain, the National Institutes of Health, and the University of Arizona guidelines for the care and use of laboratory animals. Antinociception was assessed in mice by the hot-plate assay, an assay known to be mediated by central receptors (16, 17). In the hot-plate assay, mice were placed

Bioconjugate Chem., Vol. 8, No. 3, 1997 435

on a 55 °C surface, and the mean time to lick the back paws or escape jump was recorded. Percent antinociception was calculated as 100% × (test latency - control latency)/(60 s - control latency), with a cutoff latency of 20 s (baseline). Data are presented as the mean ( SEM for groups of 10 mice. Regression lines, ED50 and AD50 values, and their 95% confidence limits were calculated using individual data points. Brain and Serum Stability Incubations. Aliquots (180 µL) of resuspended, twice washed 15% mouse brain homogenates or mouse serum were placed into 1.5-mL centrifuge tubes. The tubes were prepared in triplicate for each time point (0, 10, 15, 30, 60, 120, and 240 min). An additional set of triplicate tubes were incubated with 50 mM Tris-HCl (pH 7.4) and served as buffer controls. Twenty microliters of 1 mM peptide triglyceride complex dissolved in methanol was added to each tube, which was agitated briefly by vortex. Incubation was begun immediately at 37 °C in a rolling water-bath incubator. Enzyme activity was terminated at the end of the each incubation by adding 200 µL of CH3CN and placing the tube on ice. Each tube was centrifuged at 3000g, and 300 µL of the supernatent was transferred to a clean 1.5mL tube. An equal volume of H2O was added, and the sample was mixed for HPLC analysis. Synthetic Methods. Thin layer chromatography (TLC) was done on silica gel G plates using the following solvent systems: (A) 1-butanol/acetic acid/pyridine/water (5:5:1:4); (B) ammonium hydroxide/water/2-propanol (1: 1:3); (C) ethyl acetate/acetic acid/pyridine/water (5:5:1: 4). The compounds were visualized on the TLC plate using UV light and iodine vapor. Analytical highperformance liquid chromatography (HPLC) was performed on a Hewlett Packard 1090 II instrument with a reversed-phase C4 or C18 Vydac column eluted with a linear gradient 0-90% CH3CN (0.1% TFA) in 50 min. HPLC k′ ) [(compound retention time - solvent retention time)/solvent retention time]. 1H and 13C NMR spectra were recorded on a Varian Gemini 200 or Bruker AM 500 spectrometer in CDCl3 unless otherwise noted. Mass spectra were obtained on a MALDI Nicolet FT-MS with a CO2 laser excitation source and hydroxy benzoic acid as the thermal matrix. Amino acid analysis were performed on a Beckman 7300 amino acid analyzer, after acid hydrolysis with 4 M methanesulfonic acid for 24 h. 2-Hydroxy-1,3-propan-2-yl Dibenzoate (II). Propane-1,2,3-triol (I) (2.0 g) was added to benzoyl chloride (6.6 g) (2.2 equiv) and refluxed for 4 h; after cooling, the mixture was poured onto ice water (200 mL) whereupon a white precipitate formed that was filtered and washed with small portions of ice cold water. The solid was recrystallized from aqueous ethanol to yield II as long white needles (5.54 g, 85%); mp, 137.2 °C. MS, m/z 300.09821 found (m/z 300.09976 calcd); C, H, N, 67.96, 5.32, 0.00, (calcd 67.97, 5.33, 0.00); TLC, Rf (a) 0.72, (b) 0.48, (c) 0.31; HPLC, C18 k′ 2.26. 2-Keto-1,3-propanyl Dibenzoate (III). II (5.0 g) was added slowly to Jones reagent (1 M in acetone:water (1: 1), 20 mL) and stirred for 30 min. Propan-2-ol (50 mL) was added dropwise, and the reaction was stirred for 30 min. The resulting product was extracted with ethyl acetate (3 × 50 mL); the combined extracts were washed with water (3 × 100 mL), saturated NaHCO3 (3 × 100 mL), and saturated NaCl (3 × 100 mL), dried over MgSO4, and evaporated in vacuo. The resulting solid was decolorized with activated carbon and recrystallized with aqueous ethanol to yield III (3.62 g, 73%); mp, 162.6 °C; MS, m/z 298.08612 found (m/z 298.08411 calcd); C, H,N,

436 Bioconjugate Chem., Vol. 8, No. 3, 1997

68.40, 4.67, 0.00 (calcd 68.44, 4.69, 0.00); TLC, Rf (a) 0.53, (b) 0.68, (c) 0.71; HPLC, C18 k′ 5.82. 2-(N,N-Diisopropylamino)-2-hydroxy-1,3-propan2-yl Dibenzoate (IV). III (2.60 g) was dissolved in freshly distilled dry THF (50 mL) while under an inert atmosphere. Diisopropylethylamine (0.90 g, gold label, 1 equiv) was added very slowly via syringe pump (12 h). After a further 12 h of stirring, the mixture was evaporated to dryness in vacuo. The oily residue was purified by kesulghur distillation under vacuum. The desired product was obtained at 185-190 °C at 0.1 mmHg, which upon cooling gave a white waxy solid, to yield IV (1.21 g, 35%); mp, 86.2 °C; MS, m/z 399.20441 found (m/z 399.20455 calcd); C, H, N, 69.10, 7.29, 3.58 (calcd 69.14, 7.26, 3.50); TLC, Rf (a) 0.38, (b) 0.42, (c) 0.56; HPLC, C18 k′ 3.94. 2-(N,N-Diisopropylamino)-1,2,3-propanetriol (V). IV (1.15 g) dissolved in THF (50 mL) with a few drops of HCl (12 M) was subjected to hydrogenation over Pd (5%) on carbon at 30 psi for 4 h. Following safe removal of the residual hydrogen, the catalyst was filtered and the solvent was removed in vacuo. The crude product V was found to be of sufficient purity to proceed with the following reaction (0.46 g, 84%); mp, 31.5 °C; MS, m/z 191.167124 found (m/z 191.15213 calcd); C, H, N, 56.38, 10.96, 7.29 (calcd 56.40, 10.98, 7.32); TLC, Rf (a) 0.83, (b) 0.53, (c) 0.46; HPLC, C18 k′ 2.81. 2-(N,N-Diisopropylamino)-1,2,3-propan-2-yl Tris(octadecanyl benzoate ester) (VI). V (0.45 g) was dissolved in dry THF (50 mL) under a nitrogen atmosphere, isobutyl chloroformate (1.0 g, 3.1 equiv) was added, and the mixture was stirred for 30 min. Octadecanedioic acid monobenzyl ester XII (2.95 g, 3.1 equiv) dissolved in dry THF (50 mL) was added via syringe pump over 12 h. Ice water (100 mL) was added slowly; the organic layer was extracted with ethyl acetate (3 × 100 mL); the combined extracts were washed with water (3 × 100 mL), saturated NaHCO3 (3 × 100 mL), and saturated NaCl (3 × 100 mL), dried over MgSO4, and evaporated in vacuo to yield VI as a white waxy solid that was considered to be of sufficient purity to proceed with the following reaction (2.26 g, 70%); mp, 75.8 °C; MS, m/z 1349.99128 found (m/z 1349.99836 calcd); C, H, N, 74.67, 10.03, 1.02 (calcd 74.66, 10.00, 1.03); TLC, Rf (a) 0.36, (b) 0.52, (c) 0.61; HPLC, C18 k′ 6.91. 2-(N,N-Diisopropylamino)-1,2,3-propan-2-yl Tris(octadecanedioic acid monoester) (VII). VI (1.90 g) dissolved in THF (50 mL) with a few drops of HCl (12 M) was subjected to hydrogenation over Pd (5%) on carbon at 30 psi for 4 h. Following safe removal of the residual hydrogen, the catalyst was filtered and the solvent was removed in vacuo. The crude product VII was found to be of sufficient purity to proceed with the following reaction (1.25 g, 82%); mp, 53.1 °C; MS, m/z 1079.84953 found (m/z 1079.85752 calcd); C, H, N, 70.04, 10.85, 1.33 (calcd 70.00, 10.83, 1.29); TLC, Rf (a) 0.59, (b) 0.51, (c) 0.41; HPLC, C18 k′ 3.49. 2-(N,N-Diisopropylamino)-1,2,3-propan-2-yl Tris(octadecanedioic acid monoamide (arginyl(Nγ-Pmc)proline-tert-butyl ester) monoester) (VIII). Arginyl(Nγ-Pmc)proline-tert-butyl ester XIII (3.5 g, 6.2 equiv) was dissolved in acetonitrile (50 mL). VII (1.02 g), BOP reagent (2.9 g, 6.2 equiv), and DIEA (1.8 g, 9.3 equiv) were added, and the solution was stirred for 6 h. Water (50 mL) was added; the organic layer was extracted with ethyl acetate (3 × 100 mL); the combined extracts were washed with water (3 × 100 mL), saturated NaHCO3 (3 × 100 mL), and saturated NaCl (3 × 100 mL), dried over MgSO4, and evaporated in vacuo to yield VIII as a white waxy solid that was found to be of sufficient purity to

Patel et al.

proceed with the following reaction (2.41 g, 91%); mp, decomp.; MS, m/z 2805.78973 found (m/z 2805.79986 calcd); TLC, Rf (a) 0.27, (b) 0.36, (c) 0.61; C18 k′ 5.91; [R]20D 59.3° (c ) 1.20 MeOH). 2-Amino-1,2,3-propan-2-yl Tris(octadecanedioic acid monoamide (arginyl(Nγ-Pmc)proline-tert-butyl ester) monoester) (IX). VIII (2.10 g) was dissolved in freshly distilled tetrahydrofuran (50 mL) under an inert atmosphere at -78 °C. DIBAL-H (1.0 M in THF, 2.8 mL) was slowly added via a syringe pump over 12 h. The reaction was stirred for a further 12 h at room temperature. Acetone (20 mL) was slowly added to consume any unreacted reducing agent. Solvents were removed in vacuo, and the resulting solid was purified by reversedphase HPLC (C18 preparative column, 0-90% acetonitrile with 0.1% trifluoroacetic acid in 50 min) to yield IX as a white waxy solid (1.36, 67%); mp, decomp.; MS, m/z 2721.71643 found (m/z 2721.70597 calcd); TLC, Rf (a) 0.34, (b) 0.42, (c) 0.49; HPLC, C18 k′ 2.94; [R]20D 21.6° (c ) 1.45 MeOH). (Nr-(tert-Butyloxycarbonyl)tyrosyl(O-tert-butyl)D-alanyl-phenylalanyl-glutamyl(γ-tert-butyl)-valinylvalinyl-glycinamide)-1,2,3-propan-2-yl Tris(octadecanedioic acid monoamide (arginyl(Nγ-Pmc)prolinetert-butyl ester) monoester) (X). IX (1.25 g) was dissolved in acetonitrile (50 mL). NR-(tert-Butyloxycarbonyl)tyrosinyl(O-tert-butyl)- D -alanyl-phenylalanylglutamyl(γ-tert-butyl)-valinyl-valinyl-glycine (XIV) (0.65 g, 1.5 equiv), BOP reagent (0.34 g, 1.5 equiv), and DIEA (0.24 g, 2.5 equiv) were added, and the reaction was stirred at room temperature for 15 h. Water (50 mL) was added, and the organic layer was extracted with ethyl acetate (3 × 100 mL); the combined extracts were washed with water (3 × 100 mL), saturated NaHCO3 (3 × 100 mL), and saturated NaCl (3 × 100 mL), dried over MgSO4, and evaporated in vacuo. The crude material was purified by reversed-phase HPLC (C18 preparative column, 0-90% acetonitrile with 0.1% trifluoroacetic acid in 50 min) to yield X as a white waxy solid (1.24 g, 73%); mp, decomp.; MS, m/z 3699.24697 found (m/z 3699.25330 calcd); TLC, Rf (a) 0.21, (b) 0.39, (c) 0.69; HPLC, C18 k′ 4.38; [R]20D 53.6° (c ) 1.31 MeOH). (Tyrosyl-D-alanyl-phenylalanyl-glutamyl-valinylvalinyl-glycinamide)-1,2,3-propan-2-yl Tris(octadecanedioic acid monoamide (arginylproline) ester) (XI). X (1.24 g) was dissolved in dichloromethane (50 mL) and trifluoroacetic acid (50 mL) and stirred at ambient temperature for 1 h. After removal of the solvents in vacuo, the crude product was purified by reversed-phase HPLC on a C4 semi-preparative column eletuted with a gradient of 0-90% acetonitrile in 0.1% trifluoroacetic acid over 50 min. The corresponding collected fractions were pooled and lyphilized to yield XI as a white waxy solid (0.74 g, 88%); mp, decomp.; MS, m/z 2529.66891 found (m/z 2529.66533 calcd); TLC, Rf (a) 0.25, (b) 0.42, (c) 0.59; HPLC, C18 k′ 3.91; [R]20D 42.8° (c ) 1.19 MeOH). Octadecanedioic Acid Monobenzyl Ester (XII). Benzyl alcohol (1.03 g) was dissolved in freshly distilled tetrahydrofuran (50 mL) under an inert atmosphere, isobutyl chloroformate (1.43 g, 1.1 equiv) was added, and the mixture stirred for 30 min. Octadecanedioic acid (3.0 g) dissolved in dry THF (20 mL) was added slowly via a syringe pump over 12 h. Ice water (100 mL) was added, and the organic layer was extracted with ethyl acetate (3 × 100 mL); the combined extracts were washed with water (3 × 100 mL), saturated NaHCO3 (3 × 100 mL), and saturated NaCl (3 × 100 mL), dried over MgSO4, and evaporated in vacuo. Purification on flash silica (500 g) using methanol/chloroform (1:3) yielded XII as a white

Peptide Targeting across Blood−Brain Barrier

waxy solid (3.25 g, 85%); mp, 46.8 °C; MS, m/z 404.30221 found (m/z 404.29363 calcd); C, H, N, 74.28, 9.95, 0.00 (calcd 74.25, 9.90, 0.00); TLC, Rf (a) 0.39, (b) 0.48, (c) 0.62; HPLC, C18 k′ 2.51. Arginyl(Nγ-Pmc)proline-tert-butyl Ester (XIII). Proline-tert-butyl ester hydrochloride (2.0 g) was dissolved in acetonitrile (50 mL). NR(Fmoc)Arginine (NγPMC)-OH (6.0 g, 1.1 equiv), BOP reagent (4.2 g, 1.1 equiv), and DIEA (2.1 g, 3.1 equiv) were added, and the solution was stirred for 6 h. Water (50 mL) was added; the organic layer was extracted with ethyl acetate (3 × 100 mL); the combined extracts were washed with water (3 × 100 mL), saturated NaHCO3 (3 × 100 mL), and saturated NaCl (3 × 100 mL), dried over MgSO4, and evaporated in vacuo to an off white solid that was to be of sufficient purity to use without further purification. Piperidine 20% in DMF (50 mL) was added, and the reaction was stirred for 30 min at room temperature. The solvents were removed in vacuo. Water (50 mL) was added and adjusted to pH 8.5 with aqueous ammonia. The aqueous solution was extracted with ethyl acetate (3 × 100 mL); the combined extracts were washed with water (3 × 100 mL), saturated NaHCO3 (3 × 100 mL), and saturated NaCl (3 × 100 mL), dried over MgSO4, and evaporated in vacuo to yield XIII (3.6 g, 89%); mp, 84.8 °C as a white solid. MS, m/z 727.70221 found (m/z 727.69614 calcd); TLC, Rf (a) 0.50, (b) 0.43, (c) 0.30. HPLC, C18 k′ 3.10. [R]20D 37.9° (c ) 1.24 MeOH). Nr-tert-Butoxycarbonyl-tyrosyl(O-tert-butyl)-Dalanyl-phenylalanyl-glutamyl(γ-tert-butyl)-valinylvalinyl-glycine (XIV). Prepared by standard solidphase peptide chemistry employing NR-Fmoc protection, with HBTU/HOBt activation on a super acid labile Rink resin (18). Cleavage of the protected peptide was achieved using 2% trifluroacetic acid. The protected peptide was purified by size exclusion chromatography on a Sephadex G15 support employing 10% acetic acid in water as the eluting solvent. Lyophilization of pooled fraction yielded XIV (0.72 g, 71%); mp, decomp. MS, m/z 992.04596 found (m/z 992.00712 calcd); TLC, Rf (a) 0.52, (b) 0.48, (c) 0.34. HPLC, C18 k′ 6.91. [R]20D 56.1° (c ) 1.14 MeOH). 2-Amino-1,2,3-propan-2-yl Tris(octadecanyl benzoylate ester) (XV). VI (1.05 g) was dissolved in freshly distilled tetrahydrofuran (50 mL) under an inert atmosphere at -78 °C. DIBAL-H (1.0 M in THF, 1.4 mL) was slowly added via a syringe pump over 12 h. The reaction was stirred for a further 12 h at room temperature. Acetone (20 mL) was slowly added to consume any unreacted reducing agent. Solvents were removed in vacuo, and the resulting solid was purified by reversedphase HPLC (C18 preparative column, 0-90% acetonitrile with 0.1% trifluoroacetic acid in 50 min) to yield XV as a white waxy solid (0.68 g, 69%); mp, 56.2 °C; MS, m/z 1265.89947 found (m/z 1265.90446 calcd); C, H, N, 73.96, 9.72, 1.13 (calcd 73.93, 9.71, 1.10); TLC, Rf (a) 0.45, (b) 0.38, (c) 0.31; HPLC, C18 k′ 2.68. (Nr-(tert-Butyloxycarbonyl)tyrosinyl(O-tert-butyl)D-alanyl-phenylalanyl-glutamyl(γ-tert-butyl)-valinylvalinyl-glycinamide)-1,2,3-propan-2-yl Tris(octadecanyl benzoylate ester) (XVI). XV (0.62 g) was dissolved in acetonitrile (50 mL). NR-(tert-butyloxycarbonyl)tyrosyl(O-tert-butyl)-D-alanyl-phenylalanyl-glutyl(γ-tert-butyrate)-valinyl-valinyl-glycine (XIV) (0.36 g, 1.5 equiv), BOP reagent (0.17 g, 1.5 equiv), and DIEA (0.12 g, 2.5 equiv) were added, and the reaction was stirred at room temperature for 15 h. Water (50 mL) was added, and the organic layer was extracted with ethyl acetate (3 × 100 mL); the combined extracts were washed with water (3 × 100 mL), saturated NaHCO3 (3 × 100 mL), and saturated NaCl (3 × 100 mL), dried over MgSO4, and

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evaporated in vacuo. The crude material was purified by reversed-phase HPLC (C18 preparative column, 0-90% acetonitrile with 0.1% trifluroacetic acid in 50 min) to yield XVI as a white waxy solid, (0.77 g, 71%); mp, decomp.; MS, m/z 2228.30843 found (m/z 2228.33442 calcd); TLC, Rf (a) 0.21, (b) 0.45, (c) 0.68; C18 k′ 5.94. [R]20D 48.5° (c ) 1.82 MeOH). 2-(Tyrosyl-D-alanyl-phenylalanyl-glutamyl-valinylvalinyl-glycinamide)-1,2,3-propan-2-yl tris(octadecanyl benzoylate ester) (XVII). XVI (0.62 g) was dissolved in dichloromethane (50 mL) and trifluoroacetic acid (50 mL) and stirred at ambient temperature for 1 h. After removal of the solvents in vacuo, the crude product was purified by reversed-phase HPLC on a C4 semi-preparative column eletuted with a gradient of 0-90% acetonitrile in 0.1% trifluoroacetic acid over 50 min. The corresponding collected fractions were pooled and lyophilized to yield XVII as a white waxy solid (0.49 g, 88%); mp, decomp.; MS, m/z 2040.33948 found (m/z 2040.34459 calcd); TLC, Rf (a) 0.34, (b) 0.39, (c) 0.52; HPLC, C18 k′ 3.26. [R]20D 47.1° (c ) 1.04 MeOH). 2-(Tyrosyl-D-alanyl-phenylalanyl-glutamyl-valinylvalinyl-glycinamide)-1,2,3-propan-2-yl tris(octadecanedioic acid monoester) (XVIII). XVII (0.45 g) dissolved in THF (50 mL) with a few drops of HCl (12 M) was subjected to hydrogenation over Pd (5%) on carbon at 30 psi for 4 h. Following safe removal of the residual hydrogen, the catalyst was filtered. After removal of the solvents in vacuo, the crude product was purified by reversed-phase HPLC on a C4 semi-preparative column eletuted with a gradient of 0-90% acetonitrile in 0.1% trifluoroacetic acid over 50 min. The corresponding collected fractions were pooled and lyophilized to yield XVIII as a white waxy solid (0.35 g, 90%); mp, decomp.; MS, m/z 1770.20981 found (m/z 1770.20375 calcd); TLC, Rf (a) 0.41, (b) 0.34, (c) 0.28; HPLC, C18 k′ 3.10. [R]20D 51.3° (c ) 1.61 MeOH). 2-(N,N-Diisopropylamino)-1,2,3-propan-2-yl Tris(octadecanyl ester) (XIX). V (0.15 g) was dissolved in dry THF (50 mL) under a nitrogen atmosphere, isobutyl chloroformate (0.35 g, 3.1 equiv) was added, and the mixture was stirred for 30 min. Octadecaneoic acid (0.85 g, 3.1 equiv) dissolved in dry THF (50 mL) was added via syringe pump over 12 h. Ice water (100 mL) was added slowly, and the organic layer extracted with ethyl acetate (3 × 100 mL); the combined extracts were washed with water (3 × 100 mL), saturated NaHCO3 (3 × 100 mL), and saturated NaCl (3 × 100 mL), dried over MgSO4, and evaporated in vacuo to yield XIX as a white waxy solid that was considered to be of sufficient purity to proceed with the following reaction (0.50 g, 65%); mp, 74.9 °C; MS, m/z 989.94625 found (m/z 989.93498 calcd); C, H, N, 76.38, 12.46, 1.41 (calcd 76.36, 12.42, 1.41); TLC, Rf (a) 0.32, (b) 0.51, (c) 0.64; HPLC, C18 k′ 5.37. 2-Amino-1,2,3-propan-2-yl Tris(octadecanyl ester) (XX). XIX (0.50 g) was dissolved in freshly distilled tetrahydrofuran (50 mL) under an inert atmosphere at -78 °C. DIBAL-H (1.0 M in THF, 0.7 mL) was slowly added via a syringe pump over 12 h. The reaction was stirred for a further 12 h at room temperature. Acetone (20 mL) was slowly added to consume any unreacted reducing agent. Solvents were removed in vacuo, and the resulting solid was purified by reversed-phase HPLC (C18 preparative column, 0-90% acetonitrile with 0.1% trifluroacetic acid in 50 min) to yield XX as a white waxy solid (0.25 g, 56%); mp, 89.4 °C; MS, m/z 905.84681 found (m/z 905.84108 calcd); C, H, N, 75.54, 12.27, 1.56 (calcd 75.50, 12.25, 1.54); HPLC, Rf (a) 0.35, (b) 0.39, (c) 0.42; HPLC, C18 k′ 3.68.

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Figure 1. Retrosynthetic analysis of target and control molecules.

(Nr-(tert-Butyloxycarbonyl)tyrosyl(O-tert-butyl)D-alanyl-phenylalanyl-glutamyl(γ-tert-butyl)-valinylvalinyl-glycinamide)-1,2,3-propan-2-yl Tris(octadecanyl ester) (XXI). XX (0.25 g) was dissolved in acetonitrile (50 mL). NR-(tert-butyloxycarbonyl)tyrosyl(O-tert-butyl)-D-alanyl-phenylalanyl-glutamyl(γ-tert-butyl)-valinyl-valinyl-glycine (XIV) (0.13 g, 1.5 equiv), BOP reagent (0.07 g, 1.5 equiv), and DIEA (0.05 g, 2.5 equiv) were added, and the reaction was stirred at room temperature for 15 h. Water (50 mL) was added, and the organic layer was extracted with ethyl acetate (3 × 100 mL); the combined extracts were washed with water (3 × 100 mL), saturated NaHCO3 (3 × 100 mL), and saturated NaCl (3 × 100 mL), dried over MgSO4, and evaporated in vacuo. The crude material was purified by reversed-phase HPLC (C18 preparative column, 0-90% acetonitrile with 0.1% trifluoroacetic acid in 50 min) to yield XXI as a white waxy solid (0.37 g, 71%); mp, decomp.; MS, m/z 1889.42918 found (m/z 1889.43536 calcd); TLC, Rf (a) 0.31, (b) 0.45, (c) 0.56; HPLC, C18 k′ 4.29. [R]20D 72.5° (c ) 1.18 MeOH). 2-(Tyrosyl-D-alanyl-phenylalanyl-glutamyl-valinylvalinyl-glycinamide)-1,2,3-propan-2-yl Tris(octadecanyl ester) (XXII). XXI (0.30 g) was dissolved in dichloromethane (50 mL) and trifluoroacetic acid (50 mL) and stirred at ambient temperature for 1 h. After removal of the solvents in vacuo, the crude product was purified by reversed-phase HPLC on a C4 semi-preparative column eletuted with a gradient of 0-90% acetonitrile in 0.1% trifluoroacetic acid over 50 min. The corresponding collected fractions were pooled and lyophilized to yield XXII as a white waxy solid (0.20 g, 78%); mp, decomp.; MS, m/z 1680.29063 found (m/z 1680.28121 calcd); TLC, Rf (a) 0.33, (b) 0.41, (c) 0.47; HPLC, C18 k′ 3.82. [R]20D 60.2° (c ) 1.37 MeOH).

RESULTS

The choice of the carrier molecule was based on the system for which the application was to be made, i.e., the mammalian system. Whereas a monoglyceride peptide conjugate might form micelles that would be stable at mammalian body temperature, thus resulting in a circulatory loss of the peptide prodrug, a triglyceride carrier, having a far greater critical micelle concentration at mammalian body temperature, would minimize such a loss. Based on these considerations, the proposed target and control molecules were analyzed by a retrosynthetic process (Figure 1) to arrive at a suitable starting point for the synthesis. The masked peptide carrier conjugate was assembled by fragment condensation of the peptide, the carrier molecule, and the hydrophilic dipeptide mask. Condensation was achieved by classical solution phase amide bond formation of suitably protected fragments. The synthesis of the carrier molecule was accomplished as depicted in Figure 2, and the control molecules were accomplished as depicted in Figure 3. Briefly, the synthesis consisted of a series of protection/deprotection strategies while forming a quaternary substituted carbon center suitably functionalized to enable the final fragment condensations to be accomplished under mild conditions. The lipophilic tethers were attached V f VI (Figure 2) as part of the synthesis by formation of ester linkages to mono-protected dicarboxylic fatty acids; this also acted as part of the protection strategy. The control carriers were similarly formed from the glycerol template utilizing monocarboxylic fatty acids, V f XIX (Figure 3). The free acid triglycerides were protected prior to attachment of the deltorphin peptide. The peptide was attached to the carrier (IX f X, Figure 2) as a semiprotected analogue that could be deprotected using mild conditions. Following selective deprotection of the triglyceride free acids, the preformed semi-protected dipep-

Peptide Targeting across Blood−Brain Barrier

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Figure 2. Synthetic route toward the target peptide carrier complex (XI): (a) benzoyl chloride, reflux; (b) Jones reagent, dropwise; (c) THF/diisopropylamine, N2; (d) 5% Pd/CH2; (e) N2, isobutyl chloroformate, fatty acid; (f) 5% Pd/CH2; (g) Arg-Pro-OtBu/BOP/DIEA; (h) DIBAL-H, THF, N2; (i) protected peptide/BOP/DIEA; (j) TFA/DCM.

Figure 3. Synthetic route toward the control carrier complexes (XVIII and XXII): (e) N2, isobutyl chloroformate, fatty acid; (k) DIBAL-H, THF, N2; (l) protected peptide/BOP/DIEA; (m) TFA/DCM; (n) 5% Pd/CH2; (o) N2, isobutyl chloroformate, fatty acid; (p) DIBAL-H, THF, N2; (q) protected peptide/BOP/DIEA; (r) TFA/DCM.

tide was condensed onto the peptide carrier complex. A further deprotection yielded the desired final target molecule XI (Figure 2). The protected deltorphin peptide was synthesized by a solid-phase strategy on a highly acid labile resin using NR-Fmoc protection; side chain protection of the tyrosine and glutamic acid residues were

chosen so as to be stable to hydrogenation. The arginylproline dipeptide was synthesized by solution phase methods; the side chain and C-terminal protection groups were chosen so as to be removed in the final deprotection strategy of the whole complex. The target molecule was purified by reversed-phase high-pressure liquid chroma-

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Figure 4. Antinociception as observed in the hot plate assay.

tography. The purity and properties of the target molecule and of the control molecules were assessed by a combination of high-resolution MALDI mass spectrometry, HPLC, elemental analysis, 1H and 13C NMR, optical rotation, and amino acid analysis (see Experimental Procedures). The peptide carrier complex was evaluated for analgesic efficacy following i.p. administration in the mouse hot-plate test (Figure 4). This test measures the amount of time required to react to a standardized noxious stimulus. Substances that increase the reaction time are said to display antinociceptive effects, which may be interpreted as a measure of analgesia. It is important to note that the peptide carrier complex itself is inactive in the hot-plate test when given directly into the brain (not shown) but is highly potent and prolonged acting when given peripherally (i.p., Figure 4). The results depicted in Figure 4 show that the peptide carrier complex mediated analgesia is of very long duration. The rate of decline for the observed analgesia for the carrier is much slower than that observed for non-carrier mediated analgesia when deltorphin is given directly into the brain (not shown). In vitro binding and bioassays showed little or no evidence of affinity or efficacy for the peptide carrier complex XI (Figure 2) with opioid receptors in binding experiments in rat brain homogenates or in bioassay experiments using the electrically stimulated mouse vas deferens and guinea pig ileum assays respectively (>10 mM). The free deltorphin peptide of course is well known to be highly potent in these systems with values obtained corresponding well with literature values (19, 20). It is interesting to note that both control molecules (Figure 3) gave results similar to those of the target molecule in in vivo studies, but yet when tested for analgesia did not show any activity. The stability of the peptide carrier conjugate XI and the controls XVIII and XXII was determined in mouse serum (t1/2 ≈ 82, 61, and 63 min, respectively) and in the mouse brain (t1/2 ≈ 4.5, 5, and 6 h, respectively) by HPLC analysis. These results show that the masked peptide

carrier conjugate demonstrates greatly improved serum stability over the control molecules, but the mouse brain homogenates appear to have a greater degradative effect upon the masked molecule (compared to the control molecules), probably due to endopeptidase action upon the dipeptide. In the mouse brain homogenates, the t1/2 times are of such length that they do not merit problematic effects over the duration of observed analgesia. Peptidase action upon the active peptide-carrier bond appears to be far less destructive in the brain homogenates and yet is the primary degradative site in serum. Analysis of the products generated from these assays (results not shown) suggest that incubation with mouse serum initially results in cleavage of the peptide carrier bond, and only upon further prolonged incubation is the dipeptide-carrier bond hydrolyzed. DISCUSSION

The major goal of this study was to develop an approach by which a CNS efficacious bioactive peptide could be administered via an intravenous or other peripheral route and for this peptide to overcome the problems of enzymatic degradation and inability to cross the BBB into the brain. The unique approach undertaken here relied upon utilizing biophysical and physicochemical properties of the mammalian system, together with fundamental mass action principles. The fact that the conjugate we designed was not active in the MVD and GPI assays and has very weak binding to the δ or µ opioid receptors in rat brain membrane but had potent analgesic activity when given peripherally (i.p.) suggests that the approach design has validity. A basic premise of our design was that first the dipeptide mask must be removed by protease activity before or during absorption of the circulating complex at the BBB. Then this would be followed by hydrolysis of the lipid bonds by lipases during or after transmission of the unmasked peptide complex through the BBB. The fact that both of these degradation reactions must occur before the bioactive peptide is released in the brain is consistent with the relatively slow onset of analgesia, and

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perhaps can also account for the prolonged activity that occurs after peripheral administration. The apparent serum stability of the complex also suggests that the conjugates can remain in circulation for long periods of time to account for the prolonged analgesic activity observed. Whether the peptide crosses the BBB by active transport, by transcytosis, or by absorption-elimination across the lipid bilayer lies outside the scope of this present study. These questions and a careful evaluation of the mechanism(s) of release of the bioactive peptide from the complex will require the preparation and extensive examination of radiolabeled analogues of these conjugates. In the meantime, the results reported here provide an important new opportunity to further explore approaches to getting peptides or peptide conjugates that will cross the blood-brain barrier. Finally, the opioid receptors affected by the deltorphin peptides are of practical importance to medicine. It has been hypothesized that, if an effective δ-selective agonist can be developed, such drugs might produce analgesia with limited tolarance and addiction liability (1, 21). ACKNOWLEDGMENT

We thank Peg Davis, Ed Bilsky, Elizabeth Brownson, and Katherine B. Lee for their help in setting up the assays. This work was supported by a grant from the U.S. Public Health Service, National Institute of Drug Abuse, DA-06284. LITERATURE CITED (1) Hruby, V. J., and Gehrig, C. C. (1989) Recent Developments in the Design of Receptor Specific Opioid-Peptides. Med. Res. Rev. 9, 343-401. (2) Knapp, R. J., Vaughn, L. K., and Yamamura, H. I. (1995) Selective Ligands for µ and δ Opioid Receptors. The Pharmacology of Opioid Peptides (L. F. Tseng, Ed.) pp 1-27, Harwood Academic Publishers, New York. (3) Schiller, P. W. (1993) Development of Receptor-Selective Opioid Peptide Analogs as Pharmacological Tools and As Potential Drugs. Handbook of Experimental Pharmacology, Vol. 104/1(Opioids I), pp 681-710, Springer-Verlag, Berlin. (4) Bickel, U., Yoshiwawa, T., Landaw, E. M., Faull, K. F., and Pardridge, W. M. (1993) Pharmacological Effects In Vivo in Brain by Vector-Mediated Peptide Drug Delivery. Proc. Natl. Acad. Sci. U.S.A. 90, 2618-2622. (5) Bungaard, H. (1992) Prodrugs as a Means to Improve the Delivery of Peptide Drugs. Adv. Drug Delivery Rev. 8, 1-38. (6) Kreuter, J., Alyautdin, R. N., Kharkevich, D. A., and Ivanov, A. A. (1995) Passage of Peptides Through the Blood-BrainBarrier with Colloidal Polymer Particles (Nanoparticles). Brain Res. 674, 171-174. (7) Fukuta, M., Okada, H., Iinuma, S., Yanai, S., and Toguchi, H. (1994) Insulin Fragments as a Carrier for Peptide Delivery Across the Blood-Brain-Barrier. Pharm. Res. 11, 1681-1688.

(8) Sakata, A., Tamai, I., Kawazu, K., Deguchi, Y., Ohnishi, T., Saheki, A., and Tsuji, A. (1994) In-Vivo Evidence for AtpDependent and P-Glycoprotein-Mediated Transport of Cyclosporine-A at the Blood-Brain-Barrier. Biochem. Pharmacol. 48 (10), 1989-1992. (9) Pardridge, W. M. (1991) Peptide Drug Delivery to the Brain, Raven Press, New York. (10) Risau, W., Dingler, A., Albrecht, U., Dehouck, M.-P., and Cecchelli, R. (1992) Blood-Brain-Barrier Pericytes are the Main Source of Gamma-Glutamyl-Transpeptidase Activity in Brain Capillaries. J. Neurochem. 58, 667-672. (11) Vorbodt, A. W., Lossinsky, A. S., and Wisniewski, H. M. (1986) Characterization of Endothelial-Cell Transport in the Developing Mouse Blood-Brain-Barrier. Dev. Neurosci. 8, 1-13. (12) Shook, J. E., Pelton, J. T., Wire, W. S., Hirning, L. D., Hruby, V. J., and Burks, T. F. (1987) Pharmacologic Evaluation of a Cyclic Somatostatin Analog with Antagonist Activity at Mu Opioid Receptors In Vitro. J. Pharmacol. Exp. Ther. 240, 772-777. (13) Statistical Consultants. (1986) Am. Stat. 40, 52-60. (14) Hawkins, K. N., Knapp, R. J., Lui, G. K., Guyla, K., Kazmierski, W., Wan, Y.-P., Pelton, J. T., Hruby, V. J., and Yamamura, H. I. (1989) [3H]-[H-D-Phe-Cys-Tyr-D-Trp-OrnThr-Pen-Thr-NH2]([3H]CTOP). A Potent and Highly Selective Peptide for Mu-Opioid Receptors in Rat-Brain. J. Pharmacol. Exp. Ther. 248, 73-80. (15) Vaughn, L. K., Knapp, R. J., Toth, G., Wan, Y.-P., Hruby, V. J., and Yamamura, H.I. (1989) A High-Affinity, Highly Selective Ligand for the Delta Opioid Receptors[3H][D-Pen2, pCl-Phe4,D-Pen5]Enkephalin. Life Sci. 45, 1001-1008. (16) Horan, P., Mattia, A., Bilsky, E. J., Weber, S., Davis, T. P., Yamamura, H. I., Malatynsk, E., Appleyard, S. M., Slaninova, J., Misicka, A., Lipkowski, A. W., Hruby, V. J., and Porreca, F. (1993) Antinociceptive Profile of Biphalin, A Dimeric Enkephalin Analog. J. Pharmacol. Exp. Ther. 265, 1446-1454. (17) Heyman, J. S., Mulvaney, S. A., Mosberg, H. I., and Porreca, F. (1987) Opioid δ Receptor Involvement in Supraspinal and Spinal Antinociception in Mice. Brain Res. 420, 100-108. (18) Rink, H. (1987) Solid-Phase Synthesis of Protected PeptideFragments Using a Trialkoxy-Diphenyl-Methylester Resin. Tetrahedron Lett. 28, 3787-3791. (19) Erspamer, V., Melchiorri, P., Falconieri-Erspamer, G., Negri, L., Corsi, R., Sererini, C., Barra, D., Simmaco, M., and Kriel, G. (1989) Deltorphins: A Family of Naturally Occurring Peptides With High Affinity and Selectivity for δ Opioid Binding Sites. Proc. Natl. Acad. Sci. U.S.A. 86, 5188-5192. (20) Amiche, M., Sagan, S., Mor, A., Delfour, A., and Nicholas, P. (1989) Dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-AspNH2)sA Potent and Fully Specific Agonist for the DeltaOpioid Receptor. Mol. Pharmacol. 35, 774-779. (21) Rapaka, R. S., and Porecca, F. (1992) Lack of Antinociceptive Efficacy of Intracerebroventricular [D-Ala2,Glu4]Deltorphin, But Not [D-Pen2,D-Pen5]Enkephalin In the MuOpioid Receptor Deficient CxBk Mouse. Pharm. Res. 8, 1-8.

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