How to Enhance the Permeability and Water Solubility of Peptides?

Most peptides possess polar side chains, with only a few exceptions being hydrophobic peptides. Additionally, peptide bonds in peptide molecules can form hydrogen bonds with water molecules, granting most peptides excellent water solubility. However, peptide drugs must cross the cell membrane to be absorbed into the bloodstream and exert their pharmacological activity. Therefore, structural modification and optimization of peptides are necessary to enhance their permeability, facilitating their entry into cells and achieving therapeutic effects. Methods to enhance the permeability of peptide molecules include the introduction of halogen atoms, removal of polar side chains, chiral strategies, N-alkylation, higher fatty acid modifications, and other approaches.

Introduction of halogen atoms

In the chemical modification of small-molecule drugs, introducing halogen atoms is a common strategy to enhance lipophilicity. Similarly, incorporating halogen atoms in peptide molecules can increase their lipid solubility. Neuropeptide endomorphin exhibits potent analgesic activity; however, as a peptide molecule, endomorphin 57 struggles to cross the blood-brain barrier (BBB) to reach the brain and exert its effects. Drug molecules generally require a certain degree of lipophilicity to penetrate the BBB.

To address this, Wang Rui and colleagues from Lanzhou University employed a strategy of halogen atom introduction. They substituted proline at position 2 with D-alanine and introduced halogen atoms to phenylalanine at position 4 to enhance the overall lipophilicity of the peptide molecule. This strategy significantly improved the permeability of endomorphin analogs, enabling them to cross the BBB. The partition coefficient (D) of endomorphin 57 was only 12.5, but the introduction of halogen atoms increased the D value of the analogs to 120, a nearly ninefold enhancement. Animal experiments confirmed the presence of peptide 77 in brain tissue, further validating that the introduction of halogen atoms can enable peptide molecules to cross the BBB.

Introduction of halogen to improve permeability of endomorphin(PENG Jing-jing, et al. 2020)

Removal of Polar Side Chains

Peptide molecules often contain polar carboxyl group fragments, which result in poor cell permeability for peptides rich in glutamic acid and aspartic acid. To improve the properties of such peptides, a common strategy is the removal of polar side chains. This approach has several advantages:

• Size Reduction: Removing polar side chains reduces the size of peptide molecules, making them resemble small organic molecules more closely.
• Improved Permeability: This modification enhances the cellular permeability of peptides, facilitating their entry into cells to exert their therapeutic effects.

A classic example of this approach is the development of telaprevir, an antiviral drug for hepatitis C. Early research by Vertex Pharmaceuticals identified a substrate decapeptide (compound 78) with an activity of 0.89 μmol·L^-1. However, its large molecular weight necessitated optimization to reduce its size. The research team investigated the impact of removing different amino acid fragments on the antiviral activity of the compound:

• Significant Activity Impact: Removing the P4' amino acid fragment significantly affected activity.
• Minimal Impact: Removing the P2' and P3' amino acid fragments had negligible effects on the enzyme's affinity.
• Adverse Effects: Removing the P5 and P6 acidic side chain amino acid fragments significantly reduced activity, as did the removal of P3 and P4 hydrophobic amino acid fragments.

Considering the need for binding to serine protease, researchers introduced an electrophilic aldehyde group at the C-terminus as a reactive "warhead." This modification resulted in a hexapeptide aldehyde (compound 79) that spanned S6 to S1 binding sites. The activity of this smaller molecule was equivalent to that of the original decapeptide, but with a significantly reduced molecular weight.

Optimization of anti-HCV drug telaprevir(PENG Jing-jing, et al. 2020)

Optimization of anti-HCV drug telaprevir(PENG Jing-jing, et al. 2020)

Although the acidic amino acid fragments P5 and P6 are crucial for activity, the high polarity of hexapeptide aldehyde 79 due to the presence of two carboxyl groups hinders the compound's ability to enter virus-infected cells. Therefore, the next step in structural modification focuses on improving the molecule's membrane permeability. Researchers further truncated the P5 and P6 amino acid fragments and replaced them with heterocycles to obtain tetrapeptide aldehyde 19. This resulted in a significant reduction in antiviral activity; however, compared to the decapeptide substrate 78, the molecular weight was halved, enhancing the overall druggability of the molecule.

Due to the poor metabolic properties of the aldehyde group, the aldehyde warhead was replaced with other warheads, yielding ketoamide compound 20, which exhibited excellent antiviral activity and stability. This successfully addressed the issue of membrane permeability in peptide molecules.

There was still room for improvement in the activity of compound 20. Researchers systematically optimized the P1-P4 fragments and discovered that the hydrophobic group of proline greatly influenced the enzyme's affinity. They first modified the P2 fragment by comparing various hydrophobic groups, such as ethers, esters, and carbamates. Ultimately, a tetrahydroisoquinoline carbamate derivative, compound 80, was obtained, improving the inhibitory activity against NS3/4A protease to 0.22 μmol·L^-1.

Further optimization of P1 revealed that the S1 pocket could only accommodate small hydrophobic groups, with the ethyl side chain being identified as optimal. Simultaneous optimization of P3 and P4 yielded compound 81, which demonstrated activity comparable to that of compound 80. However, the cLogP (5.5) of compound 81 better adhered to Lipinski's Rule of Five, making it the focus of further research.

Cyclization of the ethyl side chain with proline in P2 was then conducted, leading to the identification of compound 82. This compound exhibited an inhibitory activity of 44 nmol·L^-1 against the hepatitis C virus NS3/4A protease, making it a highly potent serine protease inhibitor named telaprevir. Telaprevir was approved by the FDA in 2011 for the treatment of hepatitis C virus infections.

Chirality Strategy

The secondary structure of cyclic peptides is closely related to their physicochemical and pharmacological properties. Li Zigang and colleagues from Peking University Shenzhen Institute proposed a hypothesis-introducing a chiral center into the connecting chain of constrained peptides to alter the physicochemical properties and secondary structure of peptide molecules, thereby affecting their membrane permeability. To verify the rationality of this strategy, they designed and synthesized two FITC-labeled cyclic peptide compounds, 83 and 84, containing chiral centers. Due to the presence of the chiral center, peptides 83 and 84 exist as a pair of diastereomers. These diastereomers, 83a/b and 84a/b, were separated and incubated with HEK293T cells at 37°C for 2 hours. Fluorescence confocal microscopy was used for imaging. The results showed that one configuration, the diastereomers 83b and 84b, could penetrate HEK293T cells, while the other configuration, 83a and 84a, could not. This indicates that the introduction of a chiral center can alter the helical structure of the peptide, thereby affecting the membrane permeability of peptide molecules.

Introduction of chiral center in cyclopeptide linker to change permeability of peptide. (Hu K, et al)

N-Alkylation

N-alkylation of the amide bond often alters the intramolecular or intermolecular hydrogen bond interactions within peptide molecules, thereby affecting their spatial structure and subsequently changing their physicochemical properties. Intramolecular hydrogen bonds in flexible peptides are key determinants of passive diffusion. By alkylating specific amide bonds, peptide molecules can adopt the most favorable conformation to traverse the cell membrane. Beck et al. performed N-methylation on an alanine cyclic hexapeptide to examine the effect of N-methylation on the membrane permeability of the peptide. The experimental results showed that methylation of the amide nitrogen atoms at positions 1, 5; 1, 6; or 1, 2, 4, 5 significantly improved the permeability of the peptide molecules to Caco-2 cells, with permeability comparable to that of the control testosterone (a marker for cell membrane permeability). Analysis of the spatial conformation of the N-methylated cyclic hexapeptide at positions 1 and 6 revealed that the amide hydrogen at position 2 forms an intramolecular hydrogen bond with the amide carbonyl at position 5, while the β-turn formed by the amino acids at positions 3 and 4 also contributes to intramolecular hydrogen bonding. The entire molecule adopts a hydrophobic conformation, which results in enhanced cellular permeability.

N-alkylation to improve permeability of peptides. (Beck JG, et al)

Fatty Acid Modification

A common strategy to enhance the membrane permeability of peptide molecules is the modification of peptides with high-level fatty acids. Fatty acids include both unsaturated and saturated fatty acids. Some peptide drugs modified with saturated fatty acids are already on the market for the treatment of diseases or are in clinical research stages. Fatty acids are key components of phospholipid bilayers and human fat, so modifying peptides with fatty acids can improve their affinity for the cell membrane surface, thereby enhancing their membrane permeability and promoting epithelial cell absorption of peptide molecules. Hashizume et al. modified the side chains of insulin molecules with palmitic acid, which increased the lipophilicity of palmitoylated insulin. The researchers labeled insulin with isotopes and inferred its plasma concentration by measuring radioactivity in the plasma 6 hours after administration. The results showed that the concentration of dipalmitoyl insulin was six times higher than that of native insulin, while the concentration of monopalmitoyl insulin was three times higher than that of native insulin. This also indicates that high-level fatty acid modification can enhance the membrane permeability of peptide molecules.

Introduction of fatty acid to improve the permeability of insulin. (Hashizume M, et al)

Other Approaches

In addition to chemical strategies, certain formulation techniques can significantly influence the permeability and absorption of peptide compounds. One notable example is sodium N-[8-(2-hydroxybenzyl)amino]caprylate (SNAC), a macromolecule delivery technology developed by Emisphere.

Features of SNAC:

• Delivery Capability: Effective for macromolecules ranging from 0.5 to 150 kDa.
• Structural Integrity: Preserves the higher-order structure of macromolecules, ensuring drug functionality.
• Release Behavior: Does not affect drug release properties.
• Safety: Exhibits high safety, with no impact on the structural integrity of the gastrointestinal mucosa.

Mechanism of Action:

Absorption enhancers like SNAC interact with drug molecules through weak non-covalent interactions, forming temporarily stable intermediates. These enhancers are typically hydrophobic and increase the lipophilicity of the drug-enhancer complex, facilitating the transport of the drug across epithelial cell membranes.

Once the drug-enhancer complex enters the bloodstream, the weak non-covalent bonds dissociate, releasing the drug molecule. This approach provides a safe and effective method to improve the oral bioavailability and systemic delivery of peptide-based therapeutics without altering their fundamental properties.

Mechanism of absorption enhancer. (Arbit E, et al)

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Enhancing the Water Solubility of Peptide Molecules

Peptides containing hydrophobic side chains generally have poor water solubility, while those with polar side chains tend to have better solubility. Different peptides have varying solubility depending on their composition. Some clinically used peptide drugs often contain aromatic amino acids such as phenylalanine and tyrosine, but peptides containing these aromatic amino acids frequently exhibit poor solubility. Glucagon, for instance, contains more than half hydrophobic side chains and several aromatic amino acids, making it poorly soluble in aqueous solutions.

Clinically, glucagon is commonly used to treat acute hypoglycemia. The glucagon used in clinical settings is typically stored as a lyophilized powder, which must be dissolved with a sterile acidic solvent before use. However, this often results in the formation of insoluble fibers. Therefore, improving the solubility of glucagon through appropriate modification strategies is crucial for its clinical use. The natural glucagon 85 has minimal solubility in PBS. Morz et al. replaced phenylalanine or tyrosine in glucagon with pyridylalanine (3-pal or 4-pal), and particularly when multiple sites were substituted, the resulting peptide exhibited improved solubility in PBS solutions. Specifically, peptides 87 and 88 showed enhanced solubility, greater than 15 mg·mL⁻¹, while maintaining the biological activity of glucagon. This suggests that the introduction of pyridyl groups can improve the water solubility of peptide molecules. Mayer et al. also reported using pyridyl groups to replace the phenyl rings in phenylalanine or tyrosine to enhance the water solubility of peptide calcitonin gene-related peptide receptor antagonists.

References

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