Enhancement of oral drug absorption-effect of lipid conjugation on the enzymatic stability and intestinal permeability of l-Glu-l-Trp-NH(2)

The dipeptide l-Glu-l-Trp-OH (IM862) is currently under development for the treatment of certain cancers and immuno-deficiency disorders. However, due to its highly hydrophilic character, IM862 demonstrates low permeability across biological membranes, including the gastro-intestinal track, which makes it not orally available. In this study, the effect of lipid conjugation on the stability and intestinal permeability of the IM862 amide derivative l-Glu-l-Trp-NH(2) was investigated using enzymatic extracts and monolayers of Caco-2 cells, respectively. A series of eleven novel lipopeptide analogues of l-Glu-l-Trp-NH(2) was synthesized using tert-butyloxycarbonyl or 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis. In vitro assays demonstrated an improved stability to proteolytic enzymes and increased intestinal permeability for several conjugates, thereby supporting the hypothesis that lipidation may provide a means to enable the oral administration of IM862.     

Relationship between structure and permeability of dipeptide derivatives containing tryptophan and related compounds across human intestinal epithelial (Caco-2) cells

The permeability of dipeptide derivatives containing tryptophans and indole derivatives through Caco-2 cells was used as an in vitro intestinal absorption model in order to clarify structural factors which influence their intestinal epithelial permeation and metabolism. Most peptide derivatives were hydrolysed not only by the cytosolic enzymes in Caco-2 cells during permeation but also by enzymes released to the apical solution before cell permeation. The N-terminal blocked dipeptides were more resistant to hydrolases expressed in the Caco-2 cells and indole derivatives were not entirely degraded. Based on compound concentration dependency and comparison of permeability coefficients in apical-to-basolateral and basolateral-to-apical directions, the main absorption mechanism of compounds were determined. Compounds were then classified into three groups; (1) passively transported compounds, (2) actively transported compounds and (3) compounds excreted by P-glycoprotein.     

In vitro permeability and metabolic stability of bile pigments and the effects of hydrophilic and lipophilic modification of biliverdin

Bile pigments, including bilirubin and biliverdin are tetrapyrrolic, dicarboxylic acids capable of forming conjugates at their propionic acid groups via ester or amide bonds. They possess substantial antioxidant and anti-mutagenic activities and therefore their intestinal absorption might influence the development of cardiovascular disease and cancer. The aim of this study was to investigate whether altering the physico-chemical properties of bile pigments would improve their permeability in an in vitro assay of absorption. Native and synthetically modified bile pigments were tested for gastrointestinal permeability and metabolic stability using the Caco-2 cell line. In addition, a gross measure of their toxic effects was tested in a red blood cell co-incubation assay. The apparent permeability of unconjugated bilirubin (1), bilirubin ditaurate (2) and biliverdin (3) through Caco-2 cell monolayers was determined to be 10.4+/-1.2x10(-7), 35.2+/-3.4x10(-7) and 37.0+/-1.6x10(-7) cm/s (mean+/-SD), respectively, while biliverdin diglucosamine (4), and biliverdin dioctylamine (5) were impermeable. Unconjugated bilirubin, biliverdin, bilirubin ditaurate and biliverdin diglucosamine did not decompose when incubated in Caco-2 cell homogenates, whereas biliverdin dioctylamine decomposed over time. Only unconjugated bilirubin showed toxicity towards red blood cells (> or = 1000 microM), an effect that was abolished by the addition of 40 g/L serum albumin. The data presented here suggest that bile pigments are absorbed across the Caco-2 cell monolayer and that conjugation of biliverdin to hydrophilic or lipophilic moieties decreases their absorption and can reduce their metabolic stability. In summary, exogenous bilirubin and biliverdin supplements could be absorbed across the intestinal epithelium in vivo and potentially increase circulating concentrations of these antioxidant compounds.     

Significance of substrate hydrophobicity for recognition by an oligopeptide transporter (PEPT1).

Our previous paper [(1999) Bioconjugate Chem. 10, 24-31] pointed out that hydrophobicity of substrates/inhibitors plays an important role in the recognition by an oligopeptide transporter (PEPT1) expressed in the human intestinal epithelial cell line Caco-2. To determine the significance of that hydrophobicity, we have now synthesized dipeptide analogues conjugating the epsilon-amino group of Lys in Val-Lys with aliphatic carboxylic acids: acetic acid (C2), propanoic acid (C3), pentanoic acid (C5), hexanoic acid (C6), and decanoic acid (C10). The affinities of these conjugates were estimated by their inhibition of the accumulation rate of Gly-Sar, a well-established substrate for PEPT1. With the increase in length of the hydrocarbon chain of the conjugates, i.e., in the hydrophobicity of the conjugates, the inhibition strengthened. Dixon-Webb plot analysis of the inhibition by the C10-conjugated dipeptide showed competitive inhibition. The trans-stimulation effect of Val-Lys conjugated to C10 or C5 on the uptake of Ceftibuten was observed using rat brush border membrane vesicles. This findings showed that these conjugates are transportable substrates. These results confirmed that the hydrophobicity of substrates/inhibitor is one of the factors in the recognition by PEPT1.     

Improvement of enzymatic stability and intestinal permeability of deuterohemin-peptide conjugates by specific multi-site N-methylation

The deuterohemin-peptide conjugate, DhHP-6 (Dh-β-AHTVEK-NH₂), is a microperoxidase mimetic, which has demonstrated substantial benefits in vivo as a scavenger of reactive oxygen species (ROS). In this study, specific multi-site N-methylated derivatives of DhHP-6 were designed and synthesized to improve metabolic stability and intestinal absorption, which are important factors for oral delivery of therapeutic peptides and proteins. The DhHP-6 derivatives were tested for (1) scavenging potential of hydrogen peroxide (H₂O₂); (2) permeability across Caco-2 cell monolayers and everted gut sacs; and (3) enzymatic stability in serum and intestinal homogenate. The results indicated that the activities of the DhHP-6 derivatives were not influenced by N-methylation, and that tri-N-methylation of DhHP-6 could significantly increase intestinal flux, resulting in a two- to threefold higher apparent permeability coefficient. In addition, molecules with N-methylation at selected sites (e.g., Glu residue) showed high resistance against proteolytic degradation in both diluted serum and intestinal preparation, with 50- to 140-fold higher half-life values. These findings suggest that the DhHP-6 derivatives with appropriate N-methylation could retain activity levels equivalent to that of the parent peptide, while showing enhanced intestinal permeability and stability against enzymatic degradation. The tri-N-methylated peptide Dh-β-AH(Me)T(Me)V(Me)EK-NH₂ derived from this study may be developed as a promising candidate for oral administration.     

Degradation of aromatic compounds in plants grown under aseptic conditions

The aim of the work is to investigate the ability of higher plants to absorb and detoxify environmental pollutants - aromatic compounds via aromatic ring cleavage. Transformation of 14C specifically labelled benzene derivatives, [1-6-14C]-nitrobenzene, [1-6-(14)C]-aniline, [1-(14)C]- and [7-(14)C]-benzoic acid, in axenic seedlings of maize (Zea mays L.), kidney bean (Phaseolus vulgaris L.), pea (Pisum sativum L.) and pumpkin (Cucurbita pepo L.) were studied. After penetration in plants, the above xenobiotics are transformed by oxidative or reductive reactions, conjugation with cell endogenous compounds, and binding to biopolymers. The initial stage of oxidative degradation consists in hydroxylation reactions. The aromatic ring can then be cleaved and degraded into organic acids of the Krebs cycle. Ring cleavage is accompanied by 14CO2 evolution. Aromatic ring cleavage in plants has thus been demonstrated for different xenobiotics carrying different substitutions on their benzene ring. Conjugation with low molecular peptides is the main pathway of aromatic xenobiotics detoxification. Peptide conjugates are formed both by the initial xenobiotics (except nitrobenzene) and by intermediate transformation products. The chemical nature of the radioactive fragment and the amino acid composition of peptides participating in conjugation were identified.     

A concise method for the preparation of peptide and arginine-rich peptide-conjugated antisense oligonucleotide

Peptide-oligonucleotide conjugates were synthesized using two strategies: a mimetic signal peptide-conjugated oligonucleotide was assembled stepwise on CPG by using 2,2-dimethyl-3-hydroxypropionic acid as a linker. To solve the precipitation problem in the coupling reaction caused by the electrostatic interaction of arginine-rich peptides and oligonucleotide, oligonucleotides were absorbed on an anion-exchange resin, and then the on-resin fragment was applied for the conjugation with arginine-rich peptide. The peptide-antisense oligonucleotides showed permeability to the cell membrane of HepG-2 cells.     

Synthesis and assessment of first-generation polyamidoamine dendrimer prodrugs to enhance the cellular permeability of P-gp substrates

The aim of this study is to evaluate the potential use of first-generation (G1) polyamidoamine (PAMAM) dendrimers as drug carriers to enhance the permeability, hence oral absorption, of drugs that are substrates for P-glycoprotein (P-gp) efflux transporters. G1 PAMAM dendrimer-based prodrugs of the water-insoluble P-gp substrate terfenadine (Ter) were synthesized using succinic acid (suc) or succinyl-diethylene glycol (suc-deg) as a linker/spacer (to yield G1-suc-Ter and G1-suc-deg-Ter, respectively). In addition, the permeability of G1-suc-deg-Ter was enhanced by attaching two lauroyl chains (L) to the dendrimer surface (L2-G1-suc-deg-Ter). All of the G1 dendrimer-terfenadine prodrugs were more hydrophilic than the parent drug, as evaluated by drug partitioning between 1-octanol and phosphate buffer at pH 7.4 (log K(app)). The influence of the dendrimer prodrugs on the integrity and viability of human Caucasian colon adenocarcinoma cells (Caco-2) was determined by measuring the transepithelial electrical resistance (TEER) and leakage of lactate dehydrogenase (LDH) enzyme, respectively. The LDH assay indicated that the dendrimer prodrugs had no impact on the viability of Caco-2 cells up to a concentration of 1 mM. However, the IC(50) of the prodrugs was lower than that of G1 PAMAM dendrimer because of the high toxicity of terfenadine. Measurements of the transport of dendrimer prodrugs across monolayers of Caco-2 cells showed an increase of the apparent permeability coefficient (P(app)) of terfenadine in both apical-to-basolateral (A --> B) and basolateral-to-apical (B --> A) directions after its conjugation to G1 PAMAM dendrimer. The A --> B P(app) of the dendrimer prodrugs was significantly greater than B --> A P(app). The surface-modified dendrimer prodrug L2-G1-suc-deg-Ter showed the highest A --> B permeability among the conjugates.     

Synthesis of enzymatically resistant nociceptin-related peptides containing a carbamic acid residue.

Nociceptin, a 17-amino acid peptide (FGGFTGARKSARKLANQ, N/OFQ), is the endogenous ligand of the nociceptin/orphanin FQ (NOP) receptor. This receptor-ligand system is involved in various physiological as well as pathophysiological mechanisms, but owing to the peptidic structure, it is rapidly degraded by enzymes. The enzymatic digestion of nociceptin involves mainly aminopeptidases and yields Noc(2-17)-OH and other smaller fragments. We aimed at increasing the enzymatic stability against aminopeptidases in the case of peptide Noc(1-13)-NH(2), which possesses the minimum sequence capable of interacting with the NOP receptor. Therefore we developed a new procedure for the synthesis of peptides with the carbamic acid residue [...-NH-CH(R)-CO-NH-CO-NH-CH(Q)-CO-.]. A set of four carbamic acid-nociceptin derivatives were produced. The carbamic acid residue was incorporated into the inner part of the peptides, building on solid phase, by using a suitable dipeptide fragment with carbamic acid residue produced by a simple and efficient three-step solution phase procedure. Enzymatic stability of carbamic acid peptides was studied in the presence of aminopeptidase M (AP-M) and in rat brain membrane homogenate. The receptor-binding properties were also studied by radioligand binding assay on crude rat brain membranes and the activity of the ligands were analyzed on isolated mouse vas deferens (MVD) tissues. We found that incorporation of the carbamic acid residue into the N-terminal part of nociceptin significantly increases the resistance against AP-M. We observed the decrease of binding affinities to the NOP receptor in case of the peptides modified in the N-terminal portion. Consequently, the incorporation of the carbamic acid residue into peptides can be proposed as a promising and reasonable tool for increasing enzymatic stability, where the native molecule is less sensitive for carbamic acid residue-related structural change.     

Caco-2 cell permeability and stability of two d-glucopyranuronamide conjugates of thyrotropin-releasing hormone

Caco-2 cell permeability and stability assays were used as an in vitro model to study the intestinal epithelial transport and stability of two analogues of thyrotropin-releasing hormone (TRH; Pyr-His-Pro-NH2). Peptide 1 (Pyr-His-Pro-D-glucopyranuronamide) was more permeable across the Caco-2 cell monolayer compared with the permeability of the parent TRH peptide (Papp=5.10+/-1.89x10(-6) cm/s c.f. Papp=0.147+/-0.0474x10(-6) cm/s respectively). The permeability of peptide 1 was improved threefold by attaching a 2-aminooctanoic acid moiety to the N-terminus to form peptide 2 (2-aminooctanoic acid-Gln-His-Pro-D-glucopyranuronamide) (Papp=16.3+/-2.47x10(-6) cm/s). The half-life for both peptide 1 and peptide 2 was approximately 20 min in a homogenate of Caco-2 cells compared with the half-life of TRH which is approximately 3 min. It was concluded that the permeability of peptides 1 and 2 was enhanced because of their increased stability, while the higher permeability of peptide 2 compared with peptide 1 may be attributed to its increased lipophilicity which results in enhanced passive diffusion.     

Synthesis of new class dipeptide analogues with improved permeability and antithrombotic activity

3-(S)-1,2,3,4-Tetrahydro-beta-carboline-3-carboxylic acid isolated from A. Chinese G. Don was found to possess moderate anti-aggregation activity, but with poor bioavailability. To improve its pharmacological property, we designed and synthesized a series of novel dipeptide analogues by incorporating tetrahydro-beta-carboline-3-carboxylic acid skeleton as an amino acid surrogate (*Trp). It turned out these dipeptide analogues exhibited good membrane permeability based on in vitro Caco-2 cell monolayers permeability assay. As a result, the overall biological properties of these molecules were significantly improved depending on the nature of the amino acid residues introduced onto the 3-position of the tetrahydro-beta-carboline moiety. It was very interesting to notice that these dipeptide analogues (5b,c,h,i,n,o,p,q) displayed a remarkable dual antiaggregatory activity in both of ADP- and PAF-induced platelet aggregation assay, and their aggregation response was significantly higher than that of aspirin (p<0.01). In addition, these dipeptide analogues were observed for the dose-dependent antithrombotic effect using in vivo rat arterial thrombosis model. The potency of antithrombotic activity of 5h,i,n,p was significantly higher than that of aspirin (n=12, p<0.01) at equal dose (5 micromol/kg).     

Recognition of modified forms of ribonuclease A by the ubiquitin system

The substrate specificity of the ubiquitin (Ub) conjugation system was explored with regard to recognition of unfolded conformation and/or oxidized methionine residues in six derivatives of bovine RNase A. Based on the following observations, ubiquitination of RNase A substrates by the enzymes in a rabbit reticulocyte extract appears to correlate with unfolded conformation rather than with methionine oxidation. 1) Methionine oxidation in already unfolded forms of RNase A does not enhance ubiquitination. 2) Fluorescence measurements and iodoacetate trapping of free sulfhydryls show that the disulfide bonds of MetSO-RNase A, in which the 4 methionine residues are oxidized to the sulfoxide, are reduced by 2 mM dithiothreitol (DTT) in standard Ub conjugation assays so that this derivative also is unfolded. 3) Although MetSO-RNase A is ubiquitinated in the absence of DTT, its intrinsic fluorescence, cation-exchange properties, and susceptibility to reduction indicate a non-native conformation. 4) Methionine sulfoxide-containing peptides that mimic regions of RNase A fail to inhibit conjugation of 125I-Ub to MetSO-RNase A. Ub adducts to two of the six derivatives (MetSO- and reduced/carboxamidomethylated MetSO-RNase A) increase when DTT is omitted from the reactions. Ubaldehyde, an inhibitor of isopeptidases that disassemble Ub-protein conjugates, increased product yields and reduced or abolished the DTT effect, suggesting that an isopeptidase specific for these two RNase A derivatives may be inactivated by oxidation. Ub conjugates of the other RNase A derivatives also increase with Ub-aldehyde but are unaffected by DTT.     

Promotion of peptide antimicrobial activity by fatty acid conjugation

Three peptides, YGAA[KKAAKAA](2) (AKK), KLFKRHLKWKII (SC4), and YG[AKAKAAKA](2) (KAK), were conjugated with lauric acid and tested for the effect on their structure, antibacterial activity, and eukaryotic cell toxicity. The conjugated AKK and SC4 peptides showed increased antimicrobial activity relative to unconjugated peptides, but the conjugated KAK peptide did not. The circular dichroism spectrum of AKK showed a significantly larger increase in its alpha-helical content in the conjugated form than peptide KAK in a solution containing phosphatidylethanolamine/phosphotidylglycerol vesicles, which mimics bacterial membranes. The KAK and AKK peptides and their corresponding fatty acid conjugates showed little change in their structure in the presence of phosphatidylcholine vesicles, which mimic the cell membrane of eukaryotic cells. The hemolytic activity of the KAK and AKK peptides and conjugates was low. However, the SC4 fatty acid conjugate showed a large increase in hemolytic activity and a corresponding increase in helical content in the presence of phosphatidylcholine vesicles. These results support the model of antimicrobial peptide hemolytic and antimicrobial activity being linked to changes in secondary structure as the peptides interact with lipid membranes. Fatty acid conjugation may improve the usefulness of peptides as antimicrobial agents by enhancing their ability to form secondary structures upon interacting with the bacterial membranes.     

Comparative detoxication. The detoxication of aromatic acids by invertebrates: detection of agmatine conjugates in scorpions

1. Benzoic acid and p-nitrobenzoic acid were converted in the scorpion Palamnaeus into N(1)-benzoylagmatine and N(1)-p-nitrobenzoylagmatine. 2. Some benzoyl- and p-nitrobenzoyl-arginine were also formed and these compounds were probable precursors of the agmatine derivatives. 3. p-Nitrobenzoyl- and p-aminobenzoyl-arginine were formed in millipedes dosed with the aromatic acids. 4. Woodlice excreted p-amino- and p-nitro-benzoic acid as their glycine conjugates and no conjugation could be found in the crab Gecarcinus and a freshwater crayfish Astacus.     

An activated triple bond linker enables 'click' attachment of peptides to oligonucleotides on solid support

A general procedure, based on a new activated alkyne linker, for the preparation of peptide-oligonucleotide conjugates (POCs) on solid support has been developed. With this linker, conjugation is effective at room temperature (RT) in millimolar concentration and submicromolar amounts. This is made possible since the use of a readily attachable activated triple bond linker enhances the Cu(I) catalyzed 1,3-dipolar cycloaddition ('click' reaction). The preferred scheme for conjugate preparation involves sequential conjugation to oligonucleotides on solid support of (i) an H-phosphonate-based aminolinker; (ii) the triple bond donor p-(N-propynoylamino)toluic acid (PATA); and (iii) azido-functionalized peptides. The method gives conversion of oligonucleotide to the POC on solid support, and only involves a single purification step after complete assembly. The synthesis is flexible and can be carried out without the need for specific automated synthesizers since it has been designed to utilize commercially available oligonucleotide and peptide derivatives on solid support or in solution. Methodology for the ready conversion of peptides into 'clickable' azidopeptides with the possibility of selecting either N-terminus or C-terminus connection also adds to the flexibility and usability of the method. Examples of synthesis of POCs include conjugates of oligonucleotides with peptides known to be membrane penetrating and nuclear localization signals.     

Dipeptide derivative synthesis catalyzed by Pseudomonas aeruginosa elastase.

Pseudomonas aeruginosa elastase was used to synthesize various N-protected dipeptide amides. The identity of the products was confirmed by FAB(+)-MS. After recrystallization, the yield of their synthesis was calculated, their purity was checked by RP-HPLC and their melting point was measured. With regard to the hydrolysis, it is well-established that the enzyme prefers hydrophobic amino acids in P'1 position and it has a wide specificity for the P1 position. This specificity was demonstrated to be quite unchanged when comparing the initial rates of peptide bond formation between different carboxyl donors (Z-aa) and nucleophiles (aa-NH2). The elastase, but not the thermolysin, was notably able to incorporate tyrosine and tryptophan in P'1 position. Furthermore, synthesis initial rates were at least 100 times faster with the elastase. To overcome the problematic condensation of some amino acids during chemical peptide synthesis, it has been previously suggested that enzymatic steps can combine with a chemical strategy. We demonstrated that the elastase readily synthesizes dipeptide derivatives containing various usual N-protecting groups. It was especially able to condense phenylalaninamide to Fmoc- and Boc-alanine. Increasing interest in peptides containing unnatural amino acids led us to try the elastase-catalyzed synthesis of Z-dipeptide amides including those amino acids in the P1 position. A synthesis was demonstrated with alphaAbu, Nle, Nva and Phg.     

Peptide Conjugates of Oligonucleotides As Enhanced Antisense Agents

The use of synthetic oligonucleotides and their analogs to block gene expression by binding the complementary RNA sequences in cells, the antisense principle, has been limited by poor uptake of the agents by cells in culture. This review describes attempts to harness by chemical conjugation the ability of certain peptides that may cross membranes to enhance the cellular uptake of oligonucleotides. These include fusogenic and hydrophobic peptides, nuclear localization signals, receptor targeting and translocating peptides, and various combinations. We also outline briefly some popular methods of peptide–oligonucleotide conjugation. Finally, we review the use of noncovalent peptide additives and the recent studies of conjugates of peptide nucleic acid (PNA) with peptides.     

Sulfate conjugation of benzo[alpha]pyrene metabolites and derivates

Sulfate conjugation of benzo[alpha]pyrene(BP) metabolites and derivatives was studied. The reaction sequence consisted of two steps; activation of sulfate ion to 3'-phosphoadenosine-5'-phosphosulfate and transfer of the activated sulfate to the BP-derivatives. Both reactions were carried out by enzymes located in the rat liver 105 000 g supernatant. The reactions required MgCl2. Phenol and quinone derivatives were generally good substrates for sulfate conjugation and different reactivities were observed with the dihydrodiol derivatives. Sulfate conjugates were more polar than their parent BP-derivatives and except for quinone conjugates were easily extracted with ethyl acetate. The role of sulfate conjugation in BP carcinogenesis is discussed.     

Synthesis of novel potential antitumor agents: 2,6-dimethoxyhydroquinone-3-mercapto-acetyl peptide conjugates.

This paper reports on an ongoing study of the use of short chain peptides as carriers of a potential anti-tumor agent: 2,6-dimethoxyhydroquinone-3-mercaptoacetic acid (DMQ-MA). In an effort to carry out anti-cancer drug design, we synthesized three new peptide-DMQ-MA conjugates: DMQ-MA-Arg-Arg-Ome, DMQ-MA-Lys(Cbz)-Arg-Ome, DMQ-MA-Lys(Cbz)-Arg-Arg-Ome; two new DMQ-MA-peptide-Chlorambucil (CRB) derivatives: DMQ-MA-Lys(CRB)-Arg-Ome, DMQ-MA-Lys(DMQ-MA)-Lys(CRB)-Arg-Ome and four tripeptide-cytotoxic agent conjugates: DMQ-MA-Lys(DMQ-MA)-Phe-Arg-Ome, DMQ-MA-Lys(DMQ-MA)-Ile-Arg-Ome, DMQ-MA-Lys(DMQ-MA)-Val-Arg-Ome, DMQ-MA-Lys(DMQ-MA)-Lys(Cbz)-Arg-Ome. These conjugates were synthesized by coupling protected amino acid residues according to Pfp/DCC methods (Pfp: Pentafluorophenol, DCC:N,N'-Dicyclohexyl-carbodiimide) in solution. After deblocking the Boc- group of the Lysine, the conjugation was achieved by reaction with the pentafluorophenyl ester of DMQ-MA in DMF. The CRB in the side chain was coupled by deblocking the lysylcarbobenzyloxy protecting group Cbz and then reacting with the pentafluorophenyl ester of Chlorambucil(CRB). Further studies on cytotoxicity and sequence specificity of DNA alkylation of these five new conjugates are being investigated.