Enhancing oral bioavailability of cyclic RGD hexa-peptides by the

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Enhancing oral bioavailability of cyclic RGD hexa-peptides by the Lipophilic Prodrug Charge Masking approach: Redirection of peptide intestinal permeability from paracellular to transcellular pathway Adi Schumacher-Klinger, Joseph Fanous, Shira Merzbach, Michael Weinmueller, Florian Reichart, Andreas F. B. Räder, Agata Gitlin-Domaglaska, Chaim Gilon, Horst Kessler, and Amnon Hoffman Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00466 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 8, 2018

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Molecular Pharmaceutics

Enhancing oral bioavailability of cyclic RGD hexapeptides by the Lipophilic Prodrug Charge Masking approach: Redirection of peptide intestinal permeability from paracellular to transcellular pathway Adi Schumacher-Klinger†, Joseph Fanous†, Shira Merzbach†, Michael Weinmüllerǂ, Florian Reichartǂ, Andreas F. B. Räderǂ, Agata Gitlin-Domagalska§, Chaim Gilon§, Horst Kesslerǂ and Amnon Hoffman*,† †

Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University

of Jerusalem, P.O. Box 12065, Jerusalem 91120, Israel. ǂ Institute for Advanced Study and Center of Integrated Protein Science, Department Chemie Technische Universität München Lichtenbergstrasse 4, 85748 Garching, Germany §

Institute of Chemistry, The Hebrew University of Jerusalem, Edmond Safra Campus, Givat

Ram Campus, The Hebrew University, Jerusalem 91904, Israel KEYWORDS: oral bioavailability, peptides, intestinal permeability, RGD integrin inhibitor, prodrug, enterocytes, metabolic stability, pharmacokinetics

ACS Paragon Plus Environment

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ABSTRACT Hydrophilic peptides constitute most of the active peptides. They mostly permeate via tight junctions (paracellular pathway) in the intestine. This permeability mechanism restricts the magnitude of their oral absorption and bioavailability. We hypothesized that concealing the hydrophilic residues of the peptide using the lipophilic prodrug charge masking approach (LPCM) can improve the bioavailability of hydrophilic peptides. To test this hypothesis, a cyclic N-Methylated hexapeptide containing the Arg-Gly-Asp (RGD) and their prodrug derivatives, masking the Arg and Asp charged side chains, were synthesized. The library was evaluated for intestinal permeability in vitro using the Caco-2 model. Further investigation of metabolic stability ex vivo models in rat plasma, brush border membrane vesicles (BBMVs) and isolated CYP3A4 microsomes and pharmacokinetic studies were performed on a selected peptide and its prodrug (peptide 12). The parent drug analogues were found to have low permeability rate in vitro, corresponding to atenolol, a marker for paracellular permeability. Moreover, palmitoyl carnitine increased the Papp of peptide 12 by 4-folds indicating on paracellular permeability. The Papp of the prodrug derivatives was much higher than their parent peptides. For instance, the Papp of the prodrug 12P was 20 folds higher as the Papp of peptide 12 in the apical to basolateral (AB) direction. Whereas the permeability in the opposite direction (BA of the Caco-2 model) was significantly faster than the Papp AB, indicating the involvement of an efflux system. These results were corroborated when verapamil, a P-gp inhibitor was added to the Caco-2 model increased the Papp AB of prodrug 12P by 3-fold. The prodrug 12P was stable in the BBMVs environment, yet degraded quickly (less than 5 min) in the plasma into the parent peptide 12. Pharmacokinetic studies in rats showed an increase in the bioavailability of peptide 12 >70 folds (from 0.58±0.11% to 43.8±14.9%) after applying the LPCM method to peptide 12 and


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converting it to the prodrug 12P. To conclude, the LPCM approach converted the absorption mechanism of the polar peptides from paracellular to transcellular pathway that affects tremendously their oral bioavailability. The LPCM method provides a solution for the poor bioavailability of RGD cyclohexapeptides and paves the way for other active hydrophilic and charged peptides with poor oral bioavailability.

1. Introduction Peptides are involved in various physiological processes,[1] thereby they open a wide range of therapy options for many diseases. Hydrophilic peptides constitute most of the peptide drugs and drug candidates. The physicochemical properties of the peptides (i.e., size, charge and lipophilicity) and their conformation (including flexibility/rigidity) direct the pathways of transport (both active and passive) via biological membranes.[2] These attributes also affect their pharmacodynamics because, in many cases, their site of action is extracellular. For instance, many G-protein coupled receptors (GPCRs) are located at the cell outer surface and comprise 60% of drug targets.[3] The significant hydrophilicity of many peptides allows their good water solubility on one hand, but limit their permeability through biological membranes, on the other.[4,5] Currently, most peptide drugs cannot be administered orally, as they do not permeate the intestinal wall.[5] Specifically, the enterocyte monolayer in the gut poses a considerable barrier for these peptide drugs and drug candidates.[6] Unlike nonpolar molecules that are absorbed through the cells (transcellularly) the polar compound typically permeates between the cells (paracellularly) through the aqueous pores created within the tight junctions (TJs). It should


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be noted that the surface area for transcellular pathway in the intestine is over 1000 times larger than that available via paracellular transport.[7] Poor metabolic stability is another limitation of therapeutic peptides. The half-life of most natural peptides is short, as they are prone to enzymatic degradation in the intestine, liver, kidney and plasma.[8,9] One constructive strategy suggested to improve the biostability is cyclization.[10–12] The most common mode is head-to-tail cyclization. It stabilizes the peptide secondary structures and restricts the different multiple peptide conformations.[13] Using this method, the cyclization of the terminal alpha amino- and carboxyl-groups (N- to C cyclization) forms a head-to-tail cyclic peptide linked by an amide bond. In the case of penta- or hexapeptides this cyclization usually requires one ᴅ-amino acid or glycine in the sequence.[14] N-methylation is yet another chemical modification, in which the amide proton is replaced by an N-methyl group. It occurs naturally in a variety of peptides including cyclosporine.[15] The Nmethylation approach is known to somewhat improve the intestinal and cellular permeability, and the pharmacological properties, including binding affinity and reduces the susceptibly to enzymatic cleavage.[16] Even though these approaches, based on chemical modification marked important advances in improving the drug-like properties of peptides, there is still a need for a technology that will enable substantial improvements in the intestinal permeability of hydrophilic peptides.[5,16] To overcome this obstacle, we hypothesized that a lipophilic prodrug charge masking (LPCM) method would elevate their membrane permeability. A prodrug is a poorly active or inactive compound, comprising the parental drug that undergoes in vivo biotransformation through chemical and/or enzymatic cleavage, enabling the delivery of the active (polar) molecule across the enterocyte monolayer barrier at efficacious levels.[17] In this work the positively charged


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guanidinium and the negatively charged carboxyl side chains of Arg and Asp respectively, were masked by lipophilic alkyl residues to create an ester and carbamate prodrug (Figure 1). The carbamate prodrug is readily bioconverted into the parent drug by ubiquitous esterases, which are present throughout the body, mainly from the carboxylesterase (CES) enzyme family.[18,19] The LPCM method allows tailoring carefully the structure of the pro-moiety and the site of bioconversion,[20] hence propose a potential approach to override the permeability difficulties while preserving the original hydrophilic properties of the active (parent) peptide when appears in the systemic blood circulation. In this report we applied the LPCM method to modify the transport route of hydrophilic peptides. The LPCM method was investigated on N-Methylated cyclic hexapeptides as a representative of active peptides that could serve for the design of other orally available peptidebased drugs. Specifically, in order to test this concept, cyclic N-methylated hexapeptide containing the Arg-Gly-Asp (RGD) motif was used as the model hydrophilic charged peptide, and a series of di-N-methylated RGD cyclic hexapeptides and their prodrug derivatives were synthesized. The RGD tripeptide sequence was discovered as a ligand for integrins, transmembranal proteins, involved in signaling between the extracellular matrix and intracellular growth processes. These proteins mediate many physiological activities, including, angiogenesis and has a potential as a drug candidate for treatment of malignancies.[21] The LPCM method was implied to the Arg and Asp charged side chains, as two hexyloxycarbonyl (Hoc) groups were linked to the guanidinium group of the Arg and a methyl ester group (OMe) was used to block the Asp side chain (12P, see Figure 1). All peptides in the library were produced by solid phase synthesis with standard Fmoc protecting strategy, as described by Weinmüller et al.[22] The library peptides differ in the position of the N-methyl groups, the ᴅ-amino acid in the


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sequence and the addition of the two Hoc and OMe groups for masking the charged Arg and Asp side chains. The main findings of this study emphasize the potential of LPCM as a chemical modification which could be utilized in the design of active peptides with improved drug-like properties, specifically the intestinal permeability following oral administration. It also illustrates the mechanism of permeation showing that once the prodrug penetrates via the transcellular pathway its permeability may be affected by influx and efflux transporters (e.g. P-glycoprotein (P-gp)) as found in this case. 2. Experimental Section 2.1.Chemistry General Experimental. Chemicals. All reagents and solvents were obtained from commercial suppliers at the highest purity available and used without further purification unless otherwise is stated. Analysis and Purification. Analytical HESI-HPLC-MS (heated electrospray ionization mass spectrometry) was performed on a LCQ Fleet (Thermo Scientific) with a connected UltiMate 3000 UHPLC focused (Dionex) on C18-columns: S1: Hypersil Gold AQ C18, 175 Å, 3 µm, 150 x 2.1 mm (for 8 or 20 minutes measurements with a flow rate of 0.7 mL/min); S2: Accucore AQ C18, 80 Å, 2.6 µm, 50 x 2.1 mm (for 5 minute measurements with a flow rate of 0.9 mL/min) (Thermo Scientific). Linear gradients (5%-95% acetonitrile content) with H2O (0.1% v/v formic acid) and acetonitrile (0.1% v/v formic acid) were used as eluents. All final compounds were analysed via analytical HPLC-MS to confirm a purity of ≥ 95% (220 nm). Semi-preparative reversed phase RP-HPLC was performed using Waters instruments: Waters 2545 (Binary Gradient Module), Waters SFO (System Fluidics Organizer), Waters 2996


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(Photodiode Array Detector), Waters 2767 (Sample Manager). It was used a Dr. Maisch C18column: Reprosil 100 C18, 5 µm, 150 x 30 mm. The Semi-preparative RP-HPLC were operated with a flow rate of 40 mL/min with a linear gradient (20 min) of H2O (+ 0.1%v/v trifluoroacetic acid (TFA)) and acetonitrile (+ 0.1% v/v TFA). Peptide Synthesis. General Procedures. Cyclic peptides were prepared according to the standard Fmoc-Solid-Phase Peptide Synthesis (Fmoc-SPPS) method using a 2-chlorotrityl chloride polystyrene (CTC) resin (100-200 mesh, max. 0.969 mmol/g) with N-Methylation of individual amino acids on the resin followed by backbone cyclization in solution as well as the removal of acid labile side chain protecting groups. The synthesis of the Hoc2-protected arginine was performed according to the previously reported method.[22] Loading of CTC-resin. Peptide synthesis was carried out using 2-chlorotrityl chloride resin (CTC-resin) (100-200 mesh, max. 0.969 mmol/g) following standard Fmoc-strategy. Fmoc-XaaOH (1.2 eq.) were attached to the CTC-resin with DIEA (2.5 eq.) in anhydrous CH2Cl2 (10 mL/g resin) at room temperature for 2 h. The remaining trityl chloride groups were capped by addition of a solution of anhydrous MeOH and DIEA (5:1; v/v; 1.0 mL/g resin) for 15 min. The resin was filtered and washed with CH2Cl2 (5x) and three times with N-methylpyrrolidon (NMP; 3x). The loading capacity was estimated to 0.969 mmol/g (100 %). On-Resin Fmoc-Deprotection. The resin-bound Fmoc peptide was treated with 20% (v/v) piperidine in dimethylformamide (DMF) for 10 minutes and a second time for 5 minutes. The resin was washed with NMP (5x). Standard Amino Acid Coupling. A solution of Fmoc-Xaa-OH (2.0 eq.), O-(7-azabenzotriazol1-yl)-N,N,N′,N′-tetramethyluronium-hexafluorphosphate

(HATU)

(2.0

eq.),

1-hydroxy-7-


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azabenzotriazole (HOAt; 2.0 eq.) and DIEA (5.0 eq.) in DMF (10 mL/g resin) was added to the resin-bound free amine peptide and shaken for 60 min at room temperature and washed with NMP (5x). On-Resin N-Methylation. The linear, Fmoc-deprotected peptide is washed with CH2Cl2 (3x) and incubated with a solution of 2-nitrobenzenesulfonyl chloride (o-Ns-Cl, 4.0 eq.) and 2,4,6collidine (10 eq.) in CH2Cl2 for 20 min at room temperature. The resin is washed with CH2Cl2 (3x) and THF abs. (5x). A solution containing PPh3 (5.0 eq.) and MeOH abs. (10 eq.) in THF abs. is added to the resin. Diisopropyl azodicarboxylate (DIAD) (5.0 eq.) in a small amount THF abs. is added stepwise to the resin and the solution is incubated for 15 min and washed with THF (5x) and NMP (5x). For o-Ns deprotection, the resin-bound o-Ns-peptides were stirred in a solution of mercaptoethanol (10 eq.) and DBU (5.0 eq.) in DMF (10 mL/g resin) for 5 minutes. The deprotection procedure was repeated one more time and the resin was washed with NMP (5x). Cleavage of Linear Peptides from the Resin. For complete cleavage from the resin the peptides were treated three times with a solution of CH2Cl2 and hexafluoroisopropanol (HFIP; 4:1; v/v) at room temperature for half an hour and the solvent evaporated under reduced pressure. Backbone Cyclization with DPPA. To a solution of peptide in DMF (1 mM peptide concentration) and NaHCO3 (5.0 eq.) diphenylphosphoryl azide (DPPA; 3.0 eq.) was added at room temperature and stirred over night or until no linear peptide could be observed by HESIMS. The solvent was evaporated in vacuo and the crude material directly used for the next step. Removal of Acid Labile Side Chain Protecting Groups. Cyclized, side chain protected peptides were stirred in a solution of TFA, CH2Cl2, water and triisopropylsilane (TIPS) (80:15:2.5:2.5;


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v/v/v) at room temperature for 1 h or until no more protected peptide could be observed by HESIMS. The removal was stopped with toluene and the mixture was coevaporated with toluene (2x) in vacuo followed by purification via HPLC. 2.2 In Vitro permeability studies Culture of colorectal adenocarcinoma 2 (Caco-2) cells. Caco-2 cells (passage 52-60, ATCC, Manassas, VA, USA) were grown in 75 cm2 flasks with approximately 0.5 × 106 cells/flask (Thermo-Fischer, Waltham, MA, USA) at 37°C in a 5% CO2 atmosphere and at relative humidity of 95%. The culture growth medium consisted of DMEM supplemented with 10% heatinactivated FBS, 1% MEM-NEAA, 2 mM L-glutamine, 1mM sodium pyruvate, 50,000 units Penicillin G Sodium and 50 mg Streptomycin Sulfate (Biological Industries, Israel). The medium was replaced every other day.[23] Caco-2 cells growth and treatment. Cells were seeded at density of 25 × 105 cells/cm2 on untreated culture inserts of polycarbonate membrane with 0.4 µm pores and surface area of 1.1 cm2. Culture inserts containing Caco-2 monolayer were placed in 12 mm transwell plates (Corning Inc, Corning, NY, USA). Culture medium was replaced every other day. Transepithelial Electrical Resistance (TEER) values were measured by Millicell ERS-2 System (Millipore, Burlington, MA, USA) a week after seeding up to experiment day (21-23 days) to ensure proliferation and differentiation of the cells. When the cells were fully differentiated and TEER values became stable (200–500 Ω·cm2). The TEER values were compared to control inserts containing only the medium. In vitro permeability studies using Caco-2 cells. The experiment was initiated by replacing the medium from both sides by apical (600 µL) and basolateral (1500 µL) buffers, both warmed to


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37°C. The Cells were incubated with the buffers solutions for 30 min at 37°C on a shaker (100 cycles/min). The apical buffer was replaced by apical buffer containing 10 µg/mL peptide derivative. 50 µL samples were taken from the apical side immediately at the beginning of the experiment, resulting in 550 µL apical volume during the experiment. Samples of 200 µL at fixed time points (20, 40, 60, 80, 100, 120 and 150 min) from the basolateral side and replaced with the same volume of fresh basolateral buffer to maintain a constant volume. The experiment included two control compounds, atenolol and metoprolol (10 µg/mL), as paracellular and transcellular permeability markers.[23] The samples were directly injected to the highperformance liquid chromatography (HPLC) system (Waters 2695 Separation Module) with a mass-spectrometer (Waters Micro-mass ZQ, Waters Corporation, Milford, MA, USA), as described in section 2.7. Caco-2 permeability study data analysis. Permeability Coefficient (Papp) for each compound was calculated from the linear plot of drug accumulated versus time, using the following equation: =

dq/dt ×

Where dq/dt is steady state appearance rate of the compound on the receiver side, C0 is the initial concentration of the drug on the donor side, and A is the exposed tissue surface area (1.1 cm2). 2.3. Metabolic Stability The Rat brush border membrane vesicles (BBMV) were prepared by Ca2+ precipitation from the combined duodenum, jejunum, and upper ileum of male rats.[24] Intestines were washed with ice cold saline and separated from mucus. The intestinal mucosa was separated from the


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luminal surface and placed immediately into buffer containing 50 nM KCl and 10mM Tris–HCl (pH 7.5, 4°C). The samples were then homogenated (Polytron PT 1200, Kinematica AG, Switzerland) and 10 mM CaCl2 was added. The homogenate was placed on a shaker for 30 min at 4°C and then centrifuged with Optima L-90K Ultracentrifuge (Beckman Coulter, Brea, CA, USA) at 10,000 g for 10 min. The supernatant was separated and centrifuged at 48,000 g for 30 min and an additional two purification steps were undertaken by suspending the pellet in 300 mM mannitol and 10 mM Hepes/Tris (pH 7.5) and centrifuging at 24,000 g for 60 min. The quality of the BBMV purification was tested using the brush border membrane enzyme markers gamma-glutamyl transpeptidase (GGT), leucine amino peptidase (LAP) and alkaline phosphatase (Sigma-Aldrich, St Louise, MO). Peptides were mixed with purified BBMVs in MES buffer (2(N-morpholino)ethanesulfonic acid, 50 mM pH 7.4) and incubated at 37°C for 90 min. Triplicate samples were taken at time 0 and after 5, 15, 30, 45, 60 and 90 min. For the plasma stability studies, 12 and 12P peptides (10 µg/mL) were mixed with fresh plasma from male Wistar rats (Harlan, Israel) and incubated at 37°C for 60 min. Triplicate samples were taken at time 0 and after 5, 10, 15, 30 and 60 min. 50 µL samples were withdrawn and the reaction was terminated by adding 100 µL ice cold acetonitrile (ACN), and further processed as described in section 2.7. 2.4.Enzymatic Inhibition Studies For determination of enzymatic inhibition by the self nano emulsifying drug delivery system (SNEDDS) or ketoconazole, pooled rat CYP3A4 microsomes (BD Biosciences, Woburn, MA, USA) were used. The reaction was initiated by adding ice cold microsomes (0.5 mg/mL final concentration) to preheated phosphate buffer (0.1M, pH 7.4) containing NADPH (0.66 mg/mL) and dispersed 12P-SNEDDS (2.8 µL, equivalent to 12 P 1 µM), with ketoconazole (3 µM) or 12P alone (1 µM). At predetermined times (0, 15 and 30 min), 50 µL samples were withdrawn


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and the reaction was terminated by adding 100 µL ice cold ACN, and further processed as described in section 2.7. 2.5.In vivo studies Male Wistar rats (Harlan, Israel), 275–300 g in weight, were used for all surgical procedures. Animals were anesthetized for the period of surgery by intraperitoneal injection of 1 mL/kg of ketamine/xylazine solution (9:1), placed on a heated surface and maintained at 37°C (Harvard Apparatus Inc., Holliston, MA). An indwelling cannula was placed in the right jugular vein of each animal for systemic blood sampling, by a method described before.[25] The cannula was tunneled beneath the skin and exteriorized at the dorsal part of the neck. After completion of the surgical procedure, the animals were transferred to cages to recover overnight (12–18 h). During this recovery period, food, but not water, was deprived. Throughout the experiment free access to food was available 4 h post oral administration. Animals were randomly assigned to the different experimental groups. For bioavailability studies, dispersed 12P SNEDDS was freshly prepared 30 min before each experiment, by vortex-mixing of the pre-concentrate in water (1:10, v/v) pre-heated to 37°C for 30 sec. Dispersed 12P SNEDDS (5 mg/kg) was administered to the animals by oral gavage (n=3). Systemic blood samples (0.35 mL) were taken at 5 min pre-dose, 20, 40, 60, 90, 180, 240 and 360 min post-dose. To prevent dehydration equal volumes of physiological solution were administered to the rats following each withdrawal of blood sample. Plasma was separated by centrifugation (5322 g, 10 min) and stored at −20°C pending analysis. In the 12P pharmacokinetic study, the parent peptide, 12, was analytically determined. 2.6. Pharmacokinetic Analysis


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Area under the plasma concentration−time curve (AUC) was calculated by using the trapezoidal rule with extrapolation to infinity by dividing the last measured concentration by the elimination rate constant (kel). Elimination rate constant values were determined by a linear regression analysis using the last points on the logarithmic plot of the plasma concentration versus time curve. Pharmacokinetic parameters such Tmax, Cmax, clearance (CL), volume of distribution (V), and bioavailability were calculated using noncompartmental analysis. 2.7. Analytical methods Plasma or BBMVs samples were spiked with metoprolol (1.5 µg/mL) as internal standard. ACN was added to each sample (2:1) and vortex-mixed for 1 min. The samples were then centrifuged (14,635g, 10 min) and the supernatant was transferred to fresh glass tubes and evaporated to dryness (Vacuum Evaporation System, Labconco, Kansas City, MO, USA). Then the glass tubes were reconstituted in 80 µL of mobile phase, centrifuged a second time (14,635g, 10 min). The amount of the compounds was determined using highperformance liquid chromatography mass spectrometry (HPLC-MS) Waters 2695 Separation Module, equipped with Micromass ZQ detector. The resulting solution was injected (10 µl) into the HPLC system. The system was conditioned as follows: for parent drug peptides (including 12) Kinetex® 2.6 µm HILIC 100Å, 100 × 2.1 mm column (Phenomenex®, Torrance, CA, USA), an isocratic mobile phase, acetonitrile:water:ammonium acetate buffer 50 mM (70:10:20, v/v/v), and for the prodrug peptides (including 12P) Luna® (Phenomenex®) 3 µm C8 100Å, 100 × 2.0 mm column, and isocratic mobile phase ACN:water supplemented with 0.1% formic acid (70:30, v/v), flow rate of 0.2 mL/min at 25°C. Limit of quantification for all peptides and prodrugs was 25 ng/mL. 2.8.Statistical analysis


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All values are expressed as mean ± standard error of the mean (SEM) if not stated otherwise. To determine statistically significant differences among the experimental groups, t-test or oneway ANOVA, followed by Tukey's test, was used. A p value of less than 0.05 was termed significant. 3. Results A total of 8 peptides were investigated. The library includes 2 derivative peptide groups with N-Methylation scaffold of NMe(1,6) that differ in the ᴅ-amino acid in the sequence: alanine for 12 and 12P and valine for 29 and 29P. There are 4 cyclic peptides with the sequence c(rGDAAA). These two pairs of peptides differ in the N-Methylation pattern (NMe(1,5) and NMe(5,6)). As can be seen for the parent peptides 5, 12, 23 and 29 (Table 2), all compounds permeate poorly through enterocyte monolayer, with permeability coefficient values in the range of atenolol (0.31±0.08 cm/s × 106), the marker for paracellular permeability. The incorporation of the two Hoc masking groups on Arg and methyl group on Asp to form the prodrugs 5P, 12P, 23P and 29P resulted in improved permeability compared to the parent drug, by 1.5-11.7 folds. For example, the apical to the basolateral (AB) Papp AB of peptide 23 (0.61±0.09 cm/s × 106) was increased by 2.6 folds for 23P (1.77±0.55 cm/s × 106). Furthermore, the permeability mechanism of the peptide library was studied by evaluating the Papp of a compound from the apical to the basolateral (AB) membrane and Papp from the basolateral to the apical membrane (BA). The Papp BA of the prodrug peptides were significantly higher than the Papp AB, ranging between 12.7 and 42.2. For instance, the Papp BA was 36.7 higher than the Papp AB for peptide 5P (0.6±0.27 vs. 22.12±3.58 cm/s × 106). The asymmetry


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between these values (Papp AB vs. Papp BA) is measured by the efflux ratios shown in Table 2. It should be noted that efflux ratios of >2 are indication for efflux system involvement.[23] Peptide 12 and its prodrug derivative 12P were chosen to be further investigated as model compounds for this library. The ClogP of peptide 12 and prodrug 12P were calculated (ChemDraw Ultra, ver. 12) and 12P presented a substantial increase (-3.3 vs. 6.6, respectively). In order to evaluate the permeability mechanism of the library, peptide 12 was incubated in Caco-2 model with palmitoyl carnitine chloride (PC) that enhances permeability of hydrophilic compounds by effecting the tight junctions of the epithelial barrier.[26] Indeed, there was an approximate 4-fold increase of the permeability rate of peptide 12 in the presence of PC as presented in Table 3, Papp AB raised from 0.04±0.02 to 0.16±0.01 cm/s × 106 for Papp AB in the case of the presence of PC. The Papp for atenolol, the marker for paracellular permeability, also increased in the presence of PC (0.19±0.02 vs. 1.16±0.12 cm/s × 106, respectively), while the Papp of metoprolol, the marker for the transcellular pathway, did not changed significantly (4.26±0.08 vs. 4.00±0.11 cm/s x106 with PC). Peptide 12P was further examined in the Caco-2 model, in the presence of verapamil, a known P-gp inhibitor (Figure 2).[27] Verapamil is shown to increase the AB Papp by 3 folds (0.79 vs. 2.37 cm/s × 106 with verapamil). Prodrug 12P was additionally tested in the presence of PC. Figure 3 shows that the presence of PC affects the Papp values compared to verapamil which is related to the inhibition of the efflux system. There is a significant difference between the Papp of peptide 12P alone (1.64±0.15 vs. 12.52±0.20 cm/s × 106), whereas in the presence of PC, the AB and BA Papp values are similar (5.37±0.16 vs. 6.80±0.28 cm/s × 106).


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The metabolic stability in rat plasma of the prodrug 12P was compared to that of peptide 12 (Figure 4a). This ex vivo model indicates the potential metabolism of peptide 12P into the parent drug, peptide 12. As can be seen in Figure 4a, peptide 12 was relatively stable throughout the study and after an hour 87.3±5.7% of the peptide was detectible from the initial concentration, while peptide 12P degraded quickly to the parent drug (t1/2 in less than 5 min, P

Enhancing oral bioavailability of cyclic RGD hexa-peptides by the

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