How to Enhance the Pharmacokinetic Stability of Peptides?
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The basic building blocks of peptides are amino acids, and their essence is the same as that of proteins. As a result, peptide molecules are substrates for many proteases, a characteristic that severely limits the development and research of peptide-based drugs. Generally speaking, most peptide drugs cannot be taken orally because they are digested and destroyed by gastric pepsin and pancreatic proteases. Even when administered by injection, peptide drugs may still be degraded and inactivated by proteases in the blood and tissues. Therefore, the bioavailability of peptide drugs is very low, which greatly limits their use in clinical treatment. In order to mitigate or avoid protease degradation of peptides, chemical methods or other approaches must be used to modify and alter the peptide molecules to enhance their metabolic stability. This provides some ideas and references for solving the metabolic stability issues of peptides in new drug development. The main strategies for enhancing the metabolic stability of peptides include modifications with unnatural amino acids, pseudo-peptization strategies, inverse peptide strategies, cyclization strategies, as well as modifications with advanced fatty acids, protein fusion strategies, and PEGylation.
Peptide Backbone Modification
The main methods for modifying and altering the peptide backbone to enhance the metabolic stability of peptide molecules include modifications with unnatural amino acids, pseudo-peptization strategies, inverse peptide strategies, and cyclization strategies.
Natural active peptides are often composed of natural amino acids. These peptides are susceptible to degradation by proteases in the body, which shortens their half-life and reduces their therapeutic effect. This makes it difficult to develop them as drugs. β-amino acids, as unnatural amino acids, are not easily recognized and hydrolyzed by proteases in the body, and thus play an important role in the structural modification and enhancement of active peptides.
Compound 51 is a neurotensin, which acts on two subtypes of neurotensin receptors, NTSR1 and NTSR2. Neurotensin and its receptors are closely related to the regulation of pain sensation, food intake, and tumor growth. Researchers optimized the structure of neurotensin 51 and, through truncation, obtained a simplified peptide 52 (amino acids from position 8 to 13; NTSR1 Ki = 0.24 nmol·L-1; NTSR2 Ki = 1.2 nmol·L-1), which showed improved activity against both NTSR1 and NTSR2 receptors compared to neurotensin 51. However, simplified peptide 52 is easily metabolized by enzymes in the body, so its half-life is very short. To address this, researchers introduced β-amino acids, resulting in an active peptide 53. Although the activity of peptide 53 against NTSR1 and NTSR2 receptors decreased (NTSR1 Ki = 8 nmol·L-1; NTSR2 Ki = 25 nmol·L-1), its half-life was extended to 32 hours. Researchers then replaced the arginine at the N-terminus with β-arginine to create peptide 54. Compared to 53, peptide 54 showed slightly improved activity (NTSR1 Ki = 6 nmol·L-1; NTSR2 Ki = 12 nmol·L-1), and its half-life was greater than 7 days. This greatly enhanced the residence time of the active peptide in the body and improved its pharmacokinetic stability.
Introduction of beta-amino acids to improve the metabolic stability of peptides(PENG Jing-jing, et al. 2020)
Peptide 55 is a widely studied inhibitor of the metalloprotease EP24.15 (endopeptidase). EP24.15 is closely linked to the regulation of pituitary function by the hypothalamus and blood pressure regulation. Literature reports suggest that EP24.15 may also be associated with the aggregation of Aβ protein and Alzheimer's disease (AD), making EP24.15 a research hotspot in psychiatric disorders. Although peptide small molecule 55 exhibits strong inhibitory activity against EP24.15 (IC50 = 0.06 μmol·L-1), it is susceptible to hydrolysis by the protease neutral endopeptidase EP24.11, which is related to EP24.15. Therefore, the primary research and development goal is to enhance the stability of 55 against neutral endopeptidase EP24.11. Researchers attempted to replace the alanine, tyrosine, and carboxyl-terminal residues in 55 with β-alanine, β-phenylalanine, and β-amino propionic acid, respectively, resulting in β-peptide 56. While the inhibitory activity of β-peptide 56 against EP24.15 decreased (IC50 = 2.8 μmol·L-1), its stability against neutral endopeptidase EP24.11 significantly improved, showing almost no degradation.
Modification of 55 with β-amino acids to yield an inhibitor with complete stability against EP24.11(PENG Jing-jing, et al. 2020)
Researchers explained the enhanced stability of peptide molecules against neutral endopeptidase by introducing β-amino acids, as illustrated in the schematic diagram. For natural α-peptides, at the specific protease cleavage site, a water molecule first forms a hydrogen bond with the amide bond, which facilitates the attack of the water molecule on the amide bond, ultimately leading to the cleavage of the amide bond. In the case of β-peptides, however, the addition of a methylene group causes a conformational change in the overall peptide structure. As a result, the water molecule at the protease cleavage site is unable to form a hydrogen bond with the amide bond, which hinders the protease from cleaving the amide bond. Therefore, β-peptides exhibit stronger resistance to hydrolysis than α-peptides.
Schematic diagram of how a β-peptide may not be cleaved by the peptidase(PENG Jing-jing, et al. 2020)
Opioid receptors are closely related to pain and include several subtypes, such as the μ, δ, and κ receptors. Opioid peptides are endogenous neurotransmitters that exert pharmacological effects by binding to these receptors. Researchers found that enkephalin 57 is an endogenous substrate peptide for the μ-receptor and exhibits strong pharmacological activity, with an agonistic activity against the μ-receptor of 14.40 nmol·L-1. Unlike morphine, it does not cause severe adverse effects. Furthermore, at effective doses, enkephalin is less likely to induce respiratory depression and cardiovascular issues. As a result, enkephalin has attracted widespread scientific attention. However, enkephalin still faces some challenges, one of which is its poor metabolic stability, with a half-life of only 16.9 minutes.
Introduction of unnatural amino acids to improve the stability of endomorphin and its potent analogues(PENG Jing-jing, et al. 2020)
The team led by Wang Rui at Lanzhou University discovered that enkephalin analogs containing unnatural amino acids have significantly improved metabolic stability and can further enhance the activity of enkephalin against the μ-receptor. They first replaced the C-terminal phenylalanine with an unnatural amino acid, resulting in compound 58, which exhibited an agonistic activity of 0.0334 nmol·L-1 against the μ-receptor, 430 times more potent than enkephalin 57. Additionally, the half-life of this compound in the brain homogenate was extended to 85.9 minutes, nearly four times longer than that of enkephalin. Subsequently, based on this work, they further replaced the tyrosine and proline segments with unnatural amino acids to produce compound 59, which showed even greater activity against the μ-receptor, reaching 0.0420 pmol·L-1. Moreover, the half-life of compound 59 in brain homogenate exceeded 600 minutes, solving the problem of the short half-life of endogenous enkephalin. Therefore, the introduction of unnatural amino acids is of significant importance in improving the metabolic stability of peptide compounds.
Natural peptides are mostly composed of L-amino acids and are easily degraded by various proteases, losing their activity. Proteolytic hydrolysis reactions are generally stereospecific. Introducing D-amino acids changes the conformation of the peptide, making it less susceptible to proteolytic degradation. Therefore, peptides modified with D-amino acids are more resistant to protease degradation.
Luteinizing hormone-releasing hormone (LHRH) is a decapeptide secreted by the hypothalamus that regulates reproductive function. It binds to the luteinizing hormone-releasing hormone receptor (GnRHR) on the anterior pituitary, regulating the synthesis and secretion of luteinizing hormone. In addition, LHRH, together with other growth factors, regulates tumor cell growth in various human malignancies. LHRH and its analogs can inhibit the proliferation of hormone-dependent tumor cells by suppressing the function of the pituitary-gonadal axis. Therefore, LHRH and its analogs are currently used in clinical settings to treat hormone-dependent tumors, such as prostate cancer and breast cancer. However, the peptide bonds between the 5th and 6th residues, as well as the 6th and 7th residues of natural LHRH, are relatively unstable and are easily cleaved by endopeptidases in the body, resulting in a half-life of only 2-4 minutes. To improve the stability of LHRH in the body, researchers have attempted to introduce various types of D-amino acids at position 6, leading to the development of marketed drugs such as Nafarelin (60) and Triptorelin (61), whose half-lives have been significantly extended to 3 hours and 4 hours, respectively.
Introduction of D amino acids to improve the stability of peptides(PENG Jing-jing, et al. 2020)
Pseudo-peptide Strategy
The peptide bond (-CONH2-) is a characteristic feature of peptide molecules, but it is easily recognized and degraded by proteases in the body, which is one of the reasons for the poor stability of peptide molecules. Pseudo-peptides involve substituting one or more atoms in the peptide bond with other atoms based on principles such as bioelectronic repulsion. Since pseudo-peptides fundamentally alter the chemical structure of the amide bond, differing from the homologous structures of proteins or peptides, they can avoid recognition and hydrolysis by proteases in the body, thus improving the stability and activity of peptide molecules.
The protein-protein interaction between the N-methyl-D-aspartate (NMDA) receptor and its intracellular postsynaptic density protein-95 (PSD-95) is a potential strategy for treating ischemic brain diseases, neuropathic pain, and Alzheimer's disease. Bach et al. reported that an N-alkylated glutamic acid-threonine-alanine-valine tetrapeptide compound (N-methyl-ETAV, 62) is an inhibitor of NMDA/PSD-95 protein-protein interaction (Ki = 9.65 μmol·L-1). Through structural modifications, they developed a series of highly active tetrapeptide derivatives. However, during the modification process, the researchers found that these compounds had poor plasma stability. For example, compound 62 had a half-life of only 113 minutes in human plasma, and its poor metabolic stability limited further development.
To improve the plasma stability of the compound, Bach and colleagues performed pseudo-peptide structural modifications by replacing the oxygen atom in the amide bond with a sulfur atom, resulting in a thioamide bond that is less susceptible to protease recognition and hydrolysis. By comparing pseudo-peptides 63 and 64, which contain thioamide bonds, with compound 62, which contains amide bonds, it was found that while the activity of the thioamide-containing compounds decreased somewhat, their plasma stability significantly increased. Notably, compound 63 showed a 50-fold increase in plasma half-life, with its activity remaining nearly unchanged (Ki = 10.8 μmol·L-1). The results indicated that thioamide-based pseudo-peptide modifications are an effective strategy for improving the plasma stability of peptide compounds.
Thionamide pseudopeptides to improve stability of peptides(PENG Jing-jing, et al. 2020)
Inverse-peptide Strategy
Proteins, hormones, active peptides, and natural product peptides are substrates for various proteases, and as such, they tend to be easily degraded by proteases and have relatively short half-lives. In addition to the strategies mentioned previously, which effectively resist proteolytic hydrolysis, changing the direction of the peptide bond can also alter the protease's recognition of the substrate, thereby providing protection against degradation. This peptide bond direction-changing strategy is called retro-peptide modification, and the resulting peptides are referred to as retro-peptides or retro-inverse peptides.
The formation of amyloid β-protein (Aβ) deposits may be an important process in the onset of Alzheimer's disease (AD). Studies have shown that soluble Aβ oligomers are cytotoxic and have potential impacts on memory and learning abilities in the brain. Inhibiting Aβ aggregation in the early stages could effectively treat AD. Taylor et al. reported a nonapeptide (65) that effectively inhibits Aβ aggregation. Despite its strong inhibitory effect on Aβ oligomer aggregation, peptide 65 has several hydrolytic cleavage sites, requiring structural modification to enhance its metabolic stability. Retro-peptide modification of 65 yielded retro-peptide 66. Theoretically, the retro-peptide maintains a similar three-dimensional structure to 65, thus preserving its activity. Experimental results also indicated that the retro-peptide 66 did not show significant changes in its inhibitory activity against Aβ oligomer aggregation.
Taylor et al. evaluated the metabolic stability of peptides 65 and 66 using protein degradation experiments. The peptides were incubated with human plasma or brain extracts for 24 hours, and the amount of intact peptide was measured using high-performance liquid chromatography (HPLC). The results showed that the concentration of retro-peptide 66 in both plasma and brain extracts was significantly higher than that of 65, with the concentration of 65 approaching 100%, indicating that retro-peptide modification can significantly enhance the metabolic stability of the compound.
Inverse-peptide strategy to improve stability of peptides(Taylor M, et al)
Peptide deformylase (PDF) is an important enzyme involved in bacterial protein biosynthesis and maturation. In both bacteria and eukaryotic organelles, protein synthesis begins with N-formylmethionine, and thus, newly synthesized peptides contain a formylated N-terminus. PDF catalyzes the removal of this formyl group from the peptide. The important role of PDF in bacterial cells makes it a novel target for designing new antibiotics to treat drug-resistant pathogens.
Cyclization strategy to improve stability of peptides(Hu XB, et al)
Building on previous work, researchers discovered that compound 67 exhibited certain antibacterial activity, with an inhibition constant (Ki) value of 92 nmol·L-1 against Escherichia coli PDF. However, compound 67 is prone to degradation by serine proteases in rat plasma, leading to its inactivation. As shown the Figure, about 25% of 67 was degraded after 5 hours of incubation in rat plasma. To enhance its plasma stability, the researchers cyclized the P1' and P3' positions and designed and synthesized the cyclic peptide analog 68. The results showed that, compared to 67, the cyclic peptide analog 68 demonstrated improved antibacterial activity, with a Ki value of 74 nmol·L-1. Additionally, its plasma stability was significantly enhanced, as incubation of 68 with rat plasma for 5 hours resulted in almost no degradation.
The α-helix is a secondary structural feature found in most peptide molecules. However, synthetic peptides often do not maintain a stable α-helix structure in aqueous solutions. To address this, researchers have developed a method to stabilize the α-helix structure of peptides using carbon-carbon bonds or other connecting chains as supporting scaffolds. Peptides produced using this approach are known as "stapled peptides," which essentially belong to a type of cyclization modification strategy. Linear peptides are flexible, and in their extended conformations, they are prone to exposing more enzymatic cleavage sites, increasing the likelihood of proteolytic degradation, and thus lowering their stability. Stapling peptides can constrain the conformation of linear peptides, reducing the probability of degradation.
The β-catenin-B-cell lymphoma 9 (BCL9) protein-protein interaction is crucial for the transcriptional activity of β-catenin. This interaction is mediated by a helix segment of 25 residues in the BCL9 protein and the binding groove of β-catenin. Shaomeng Wang et al. discovered that the mutated BCL9 peptide at position 372 (peptide 69) exhibited inhibitory activity against β-catenin (Ki = 0.94 μmol·L-1). However, peptide 69 showed poor stability, degrading by 75% in cell culture medium after 1 hour. To address this, Wang et al. designed a series of structurally stable BCL9 peptides that are less prone to metabolic degradation. During the design process, they used click chemistry to create a triazole ring as a scaffold structure, synthesizing stapled peptides 70 and 71. These stapled peptides inhibited β-catenin with Ki values of 0.61 μmol·L-1 and 0.19 μmol·L-1, respectively, maintaining their activity. At the same time, the stability of the linear peptides was significantly improved, with peptides 70 and 71 degrading by only 30% and 25%, respectively, after 1 hour in cell culture medium.
Click chemistry mediated stapled peptide to improve stability of peptides(Kawamoto KA, et al)
Exogenous Modifications
Exogenous modifications to enhance the metabolic stability of peptide molecules primarily include modifications with long-chain fatty acids, protein fusion strategies, and polyethylene glycol (PEG) modifications.
Long-Chain Fatty Acid Modifications
Long-chain fatty acid modification refers to the chemical introduction of long-chain fatty acids at specific sites of peptide drugs via covalent bonds, in order to improve the properties of peptide drugs and extend their half-life. Long-chain fatty acid modifications are generally considered to stabilize the structure and increase the stability of peptides, thereby extending the half-life of peptide drugs in the body. Additionally, long-chain fatty acids have a similar structure to the phospholipids on the surface of cell membranes. Therefore, peptides modified with fatty acids often exhibit enhanced lipophilicity, which improves their absorption in the intestines and permeation through mucosal membranes. Furthermore, long-chain fatty acids can bind to human serum albumin (HSA), and the resulting complex, being larger in molecular size, is less easily transported, thus prolonging the circulation time of the peptide in the body.
At present, research into long-chain fatty acid modifications as structural modifications is still relatively slow. However, as endogenous substances in the body, long-chain fatty acids continue to attract widespread attention from researchers. For example, Bivalirudin, derived from simplifying the structure of hirudin, is an anticoagulant developed by The Medicines Company and was approved by the FDA in December 2000. It is used as an anticoagulant in the treatment of unstable angina during percutaneous transluminal coronary angioplasty (PTCA) and percutaneous coronary intervention (PCI). However, as a peptide drug, Bivalirudin has a low exposure in the body (AUC0-t = 23.7 nmol·min·mL-1) and a short half-life (t1/2 = 15.1 min), resulting in poor pharmacokinetic properties. Before PCI, it must be administered via intravenous injection and followed by continuous intravenous infusion until the surgery is completed, which results in poor patient compliance.
To address this shortcoming, researchers chemically modified a Bivalirudin analog (73) using long-chain fatty acids to modify the amino acid side chains. When comparing peptides 73 and 74, their pharmacological activities were essentially unchanged. However, the peptide 74, modified with long-chain fatty acids, showed significant improvements in exposure (AUC0-t = 1371.7 nmol·min·mL-1) and half-life (t1/2 = 212.2 min) compared to the unmodified peptide 73 (AUC0-t = 25.7 nmol·min·mL-1, t1/2 = 13.5 min). The exposure and half-life were improved 58-fold and 14-fold, respectively.
Introduction of fatty acid to improve the pharmacokinetic properties of bivalirudin analogs(PENG Jing-jing, et al. 2020)
The marketed antidiabetic peptide drugs liraglutide and semaglutide also utilize long-chain fatty acid modifications. The incorporation of long-chain fatty acids enhances the hydrophobicity of these drugs, conceals the binding sites for dipeptidyl peptidase-4 (DPP-4), reduces renal excretion, and extends their half-life.
Liraglutide is a long-acting GLP-1 receptor agonist developed by Novo Nordisk. It shares 97% amino acid sequence similarity with native GLP-1, with only one substitution at position 34, replacing lysine with arginine. Additionally, at position 26, a 16-carbon palmitic acid side chain is introduced via a linker made of glutamic acid. After subcutaneous injection, liraglutide forms a stable heptamer at the injection site, which is slowly absorbed by subcutaneous tissues. The long-chain fatty acid modification also conceals the DPP-4 binding site and enables liraglutide to form a reversible complex with human serum albumin, significantly prolonging its absorption time in the body. This results in an extended half-life for the peptide drug. In contrast, the half-life of natural GLP-1 is very short, approximately 2 minutes. However, the palmitic acid-modified liraglutide has a half-life extended to 13 hours, which is 390 times longer.
Semaglutide, another GLP-1 receptor agonist, is a modified form of GLP-1(7-37). In semaglutide, alanine at position 8 is replaced with amino isobutyric acid, and lysine at position 34 is replaced with arginine. Additionally, at position 26, a side chain of stearic acid is introduced via a glutamic acid linker. This modification also increases the drug's hydrophobicity. Moreover, semaglutide undergoes short-chain polyethylene glycol (PEG) modification, which further enhances its stability and extends its half-life to approximately one week. This extended half-life significantly improves the dosing convenience and therapeutic efficacy of semaglutide.
Introduction of fatty acid to improve the half-life of GLP-1 analogues(PENG Jing-jing, et al. 2020)
Both liraglutide and semaglutide exemplify how fatty acid and PEG modifications can substantially enhance the pharmacokinetic properties of peptide drugs, leading to better therapeutic outcomes for chronic conditions like type 2 diabetes.
In addition to reversible binding to human serum albumin (HSA), covalent irreversible binding strategies are also commonly employed in the modification of peptide drugs.
Albuvirtide is the world's first long-acting anti-HIV-1 drug, developed by Frontier Biotechnologies Inc. Its structure, shown in Figure 19, was designed to address the limitations of enfuvirtide, the first FDA-approved HIV-1 fusion inhibitor. Enfuvirtide (75), as a peptide drug, has a relatively short half-life of 3.5 to 4.4 hours in the human body, requiring twice-daily injections, which leads to poor patient adherence.
To overcome this limitation, Frontier Biotechnologies developed albuvirtide (76), a modified version of enfuvirtide. In this design, a 3-maleimido-propionic acid (MPA) group was introduced at the lysine side chain of position 13 in the peptide sequence. MPA is capable of forming a fast and irreversible covalent bond with the thiol group of human serum albumin (HSA). This covalent modification significantly enhances the stability and half-life of the peptide in the body, reducing the injection frequency to once a week instead of twice daily, greatly improving patient compliance.
Introduction of MPA to improve the half-life of anti-HIV-1 drug(PENG Jing-jing, et al. 2020)
Protein Fusion Strategy
The protein fusion strategy involves the use of genetic engineering techniques to fuse proteins or peptides with immunoglobulin Fc fragments or human serum albumin (HSA), resulting in novel modified molecules. By fusing Fc or HSA fragments to peptide drugs, the molecular size is significantly increased, which reduces the renal clearance of the peptide drugs and thus extends their half-life.
For example, dulaglutide, a long-acting glucose-lowering drug developed by Eli Lilly, is created by fusing GLP-1 with the IgG4 (Fc) fragment. The resulting fusion peptide has a biological half-life greater than 90 hours, and its therapeutic efficacy is comparable to liraglutide. In the first three quarters of 2019, dulaglutide's sales reached $2.92 billion, surpassing liraglutide ($2.45 billion in the same period).
Human serum albumin (HSA) is the most abundant protein in blood plasma, with a half-life of around 19 days. Therefore, fusing peptide drugs with HSA can significantly extend their half-life. A notable example is albighztide, developed by GlaxoSmithKline. Albighztide is the first FDA-approved drug that uses an HSA fusion strategy, and its half-life ranges from 6 to 10 days. This demonstrates that protein fusion is an effective method for extending the duration of peptide drugs.
Polyethylene glycol (PEG) is widely used in the modification of peptide drugs due to its biodegradability, low toxicity, and non-antigenic properties. PEG modification can improve the stability of peptide drugs, reduce degradation by proteases, and prevent renal filtration, thus prolonging the drug's half-life.
A well-known success of PEG modification is PEGylated interferon α, which is used to treat hepatitis B and C. Interferon α is effective in suppressing or eliminating hepatitis viruses, but it has a short half-life of 4 hours, requiring daily injections. To address this, Schering-Plough Research Institute (SPRI) introduced PEG into interferon α. The PEG-modified interferon α has a larger molecular size, which makes it less likely to be filtered by the kidneys, resulting in a half-life extension to 40 hours.
However, the introduction of PEG can also obscure the binding sites of the peptide with its receptors, reducing the antiviral activity of interferon α. Through further investigation, SPRI found that a 12 kDa PEG modification successfully extended the half-life while maintaining the peptide's antiviral activity. Therefore, it is crucial to balance the trade-off between extending the half-life and retaining the biological activity of the peptide when using PEG modification strategies.
In summary, PEG modification is an effective strategy for enhancing the stability and half-life of peptide drugs, but careful optimization is required to balance pharmacokinetic and pharmacodynamic properties.
PEG modification to improve half-life of interferon-α(PENG Jing-jing, et al. 2020)
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