Research Progress on Brain-targeted Peptide Drug Conjugates

Brain-targeted peptide-drug conjugates offer a promising strategy for overcoming the blood-brain barrier (BBB), a major challenge in CNS drug delivery. CNS diseases, including stroke, brain cancer, and neurodegenerative disorders, lack effective treatments due to the BBB's restrictive nature, which prevents most drugs from reaching the brain. The BBB is formed by a neurovascular unit with endothelial cells, astrocytes, and tight junction proteins, creating high resistance and low permeability. Additionally, ATP-binding cassette (ABC) transporters further limit drug availability by expelling compounds back into the bloodstream.

To address this, researchers have developed brain-targeted peptide-drug conjugates, consisting of a brain-targeted peptide, linker, and payload. These conjugates utilize endogenous transport mechanisms to cross the BBB and deliver therapeutics to the brain. Compared to antibody-drug conjugates (ADCs), peptide-drug conjugates offer advantages such as lower molecular weight, easier synthesis, reduced cost, better BBB penetration, and low immunogenicity. This review will explore the structures, common examples, and challenges of brain-targeted peptide-drug conjugates, highlighting their potential for precision CNS therapy.

Structure of a Brain-targeted PDC

Brain-Targeted Peptides

The targeting component of brain-targeted peptide-drug conjugates is essential for enhancing drug properties, improving selectivity, and reducing toxicity. An ideal brain-targeted peptide should have strong target binding affinity, high stability, low immunogenicity, efficient internalization, and a long plasma half-life.

Brain-targeting peptides typically utilize receptor-mediated transcytosis (RMT) to cross the BBB. For example, ANG4043, a conjugate of Angiopep-2 and trastuzumab, was developed to improve brain penetration in HER2-positive breast cancer metastases. Angiopep-2 targets low-density lipoprotein receptor-related protein 1 (LRP1), highly expressed on BBB endothelial cells, facilitating drug transport. Studies showed ANG4043 retained HER2 binding affinity, increased brain penetration, and significantly prolonged survival in a mouse model of brain metastases.

Other brain-targeting peptides include rabies virus glycopeptide (RVG), which binds γ-aminobutyric acid (GABA) and nicotinic acetylcholine receptors (nAchR) to promote CNS transport, and THR, a 12-mer peptide that enables gold nanoparticle delivery via transferrin receptors (TfR). These findings highlight the potential of peptide-drug conjugates for neuro-oncology and CNS diseases.

Cell-penetrating peptides (CPPs), typically under 30 amino acids, facilitate drug transport via adsorptive-mediated transcytosis (AMT). Their high cell permeability and low immunogenicity make them promising delivery vehicles, though they often lack selectivity. Combining CPPs with brain-targeting peptides could enhance precision drug delivery to the CNS.

Table.1 Common cell-penetrating peptide products at Creative Peptides.

For example, TAT peptides serve as transduction domains of TAT proteins, which are transcriptional activators derived from the human immunodeficiency virus (HIV-1). These peptides can be conjugated to heterologous proteins, nanoparticles, and other carriers to enhance their permeability across the BBB. Additionally, TAT peptides facilitate the transport of quantum dots into the brain parenchyma. Studies have demonstrated that conjugating TAT peptides to ciprofloxacin-loaded nanomicelles enables the micelles to cross the BBB, thereby improving their uptake by human astrocytes. Similarly, SynB1, a carrier material derived from antimicrobial peptide protein 1, has shown advantages in drug delivery across the BBB. Experimental results indicate that SynB1 significantly increases the brain concentration of morphine-6-glucuronide (M6G) without compromising BBB integrity.

Table.2 TAT peptide related products at Creative Peptides.

Ligation Groups

A perfectly peptide drug conjugate will only release the drug once it reaches the specified site, and the linker group is particularly important in this regard. That is, once the peptide-conjugated drug reaches the target, the linker group is cleaved and the drug is released in a fully active state. Therefore, the linker must be stable during cycling and preferentially cleaved at the target site to ensure that the maximum dose of the drug reaches the target. However, most linkers are cleaved from the moment they enter the bloodstream after systemic administration, and when they reach the designated site, the few remaining drugs are absorbed by the cells. Therefore, in order to avoid interfering with the binding affinity and efficacy of the peptide and its receptor, it is necessary to consider the microenvironment in which the peptide is located, such as its length, stability, release mechanism, functional group, hydrophilicity/hydrophobicity, and other characteristics. At present, the linker groups of peptide drug conjugates are divided into two categories: cleavable groups and non-cleavable groups (succinyl sulfide, oxime and triazole, etc.). Cleavable groups are further divided into three categories: pH-sensitive (acetals, ketones and carbonates), enzyme-sensitive (esterases and amideses, carbamates), and redox-sensitive (disulfide bonds).

After systemic administration of a peptide-conjugated drug, the most unstable sites are cleaved first, followed by other sites, and eventually become a mixture that includes an unmodified drug, a drug with a partially linker, or an amino acid with a linker. Although in the development of targeted therapies, cleavable linkers are preferred over non-cleavable linkers. However, non-cleavable groups have the advantage of not being cleaved by external stimuli (e.g., chemically induced stimuli) and therefore have higher stability in circulation, so they may not be cleaved in the blood like cleavable groups and release some drugs in the blood prematurely before reaching the target site. Therefore, the specific connection mode between brain-targeted peptides and drugs should be determined according to actual needs. Gliomas are the most common and deadly primary malignant brain tumors. Most treatments often fail clinically due to the intractable issues of poor drug solubility, lack of tumor selectivity, poor permeability across the BBB, and extensive intra- and inter-tumor heterogeneity. However, an extracellular matrix (ECM) glycoprotein expressed only in the CNS, called brevican (Bcan), has been found to be upregulated in gliomas and is associated with increased tumor aggressiveness and aggressiveness. Its deglycosylated subtype, known as DG-BCAN, is found only in tissue samples from human high-grade gliomas, including gliomas, and expresses tumor specificity and consistency throughout tumor tissue. This underscores its potential as a novel glioma-specific marker for the development of new targeted drugs. As a result, the researchers screened a peptide called BTP-7, which is stable in serum and can cross the BBB to specifically bind to dg-BCAN. Therefore, the polypeptide was conjugated with camptothecin (CPT) through disulfide bonds to obtain the polypeptide-conjugated drug BTP-7-CPT. The experimental results showed that the peptide-conjugated drug showed inhibitory effect on patient-derived glioma stem cells in vitro, and increased drug delivery to tumor sites in a mouse model of human-derived glioma intracranial xenograft (PDX). Moreover, the peptide-drug conjugate also enhanced tumor toxicity and prolonged animal survival compared to healthy brain tissue. Although native BTP-7 is stable in human serum for more than 12 hours, the BTP-7-CPT peptide conjugate is completely degraded within 1 hour, suggesting that the disulfide bonds in the blood are likely to have been cleaved before reaching the tumor site. Therefore, there is a need to improve the stability of the linker group of the compound in the blood.

Payload

The payload in brain-targeted peptide drug conjugates is not limited to therapeutic drugs, but can also be contrast agents. After conjugating the drug with brain-targeted peptides, the physical and chemical properties of the drug, such as solubility, selectivity and half-life, can make the drug selective, which can improve the efficacy of the original drug, reduce toxic side effects, improve solubility and increase absorption.

For example, in the treatment of glioma, traditional chemotherapy drugs such as paclitaxel (PTX), CPT or doxorubicin (DOX) are commonly used. It is mainly used to prevent the mitosis of cancer cells or promote apoptosis through various mechanisms, so as to achieve therapeutic purposes. Although this type of drug has certain efficacy, it also has certain disadvantages, such as large toxic side effects and poor solubility. Choosing the right brain-targeted peptide conjugation can solve these problems. According to this strategy, the investigators designed PTX-coupled bifunctional peptide SynB3 and PVGLIG matrix metalloproteinase-2 (MMP-2) sensitive peptide for the treatment of glioma. The experimental results show that: 1) SynB3-PVGLIG-PTX exhibits a strong affinity with MMP-2, and it can improve water solubility by polymerization to form a special structure with a positive charge. 2) SynB3-PVGLIG-PTX released PTX in a controlled manner after MMP-2 lysis, suggesting that SynB3-PVGLIG-PTX has specific cytotoxicity to glioma cells. 3) SynB3-PVGLIG-PTX can effectively inhibit the proliferation, migration and invasion of glioma cells in vitro and in vivo. In addition, the inhibition rate of SynB3-PVGLIG-PTX was significantly higher than that of the positive drugs Temozolomide (TMZ) and PTX. 4) The combined use of MMP-2-sensitive peptides and CPPs (SynB3) not only enhances BBB permeability, but also improves the glioma-targeting effect of SynB3-PVGLG-PTX, so that the drug has high anti-tumor activity with low adverse reactions during treatment. In addition to this, brain-targeted peptide drug conjugates can also be used to deliver small interfering RNAs (siRNAs) to tumor cells. Once targeted, siRNAs can inhibit translation and subsequent protein synthesis. This combination strategy also helps to combat drug resistance.

However, if different imaging methods can be used to image the structure and function of the disease site and determine the location and state of the lesion, the disease can be diagnosed and treated more accurately. For example, the Food and Drug Administration (FDA) approved the first radionuclide-containing peptide conjugate drug, 111In-DTPA-Octreotide (octrescan), for the treatment of neuroendocrine tumors. However, studies have shown that octreotide scans have limited effect in treating tumors, so the drug is mainly used for diagnosis. The structure and function of the brain are more complex, and better treatment results can be achieved if the pathophysiological state can be observed by imaging it. In recent years, in order to simultaneously address the problems related to drug localization, drug release, and drug efficacy, multi-component structures that combine drugs and imaging agents on molecular or nanoscale platforms have been explored, which are called therapeutic diagnostic agents. This approach is expected to significantly improve brain diseases with poor therapeutic outcomes, such as in vivo molecular neuroimaging. Ben Woods et al. designed the [[99mTcO4] −CPepH3] complex, with the PepH3 peptide as the brain-targeting peptide, CPepH3 as the guest, and the 99mTcO4 radiolabeled as the host. Studies have shown that the complex is able to cross the BBB and has a higher brain accumulation than other peptides used for brain-targeted delivery. The CPepH3 structure is cage-like, and the cage-like scaffold can be used for orthogonal imaging using different techniques, such as the guest cage for Positron Emission Tomography (PET) and the host cage for magnetic resonance imaging (MRI). Similarly, the robustness and modularity of these supramolecular metal-based structures can be introduced into targeted fractions, such as peptides or antibodies. At present, the laboratory is exploring the realization of heterogeneous cages with different functions, while encapsulating anti-cancer drugs, such as cisplatin.

Common Brain-Targeted Peptide Drug Conjugates

ANG1005(GRN1005)

ANG1005 is formed by a molecule of Angiopep-2 polypeptide and three molecules of PTX conjugated through cleavable succinyl ester, Angiopep-2 targets LRP-1 and makes it cross the blood-cerebrospinal fluid barrier (BCB) and BBB, enter tumor cells, and lyse PTX in tumor cells to exert its antitumor activity. In situ cerebral perfusion experiments showed that ANG1005 entered the brain in greater amounts than PTX and could bypass P-glycoprotein (P-gp) on the BBB. In vitro experiments have shown that the anti-tumor efficacy of ANG1005 against human cancer cell lines is similar to that of PTX. In vivo experiments have demonstrated that ANG1005 inhibitory effect on human tumor xenograft is more effective than PTX, and can significantly improve the survival rate of mice implanted with U87MG glioblastoma cells or NCI-H460 lung cancer cells in the brain. In the phase I study, a single intravenous injection of the peptide drug conjugate showed that ANG1005 of therapeutic concentrations could be detected in recurrent gliomas resected 3-6 hours later, providing strong evidence for its transport and tumor penetration through BBB. In phase II clinical studies of recurrent brain metastases from breast cancer (BCBM) with or without leptomeningeal carcinomatosis subset (LMC), 77% of intracranial and 86% of extracranial patients benefited ANG1005 from intracranial injection of 600 mg/m² intravenously every 3 weeks, mainly as stable or better disease. In leptomeningeal cancer, intracranial disease was controlled in 79% of patients, and the estimated median overall survival was 8.0 months (95% confidence interval, 5.4~9.4 months). A phase III clinical trial with registration number NCT03613181 is underway to see if ANG1005 can prolong the survival of HER2-negative breast cancer patients who have recently been diagnosed with LMC and have been treated for brain metastases compared to the best drug chosen by the physician. 

DA-TP10

Parkinson's disease (PD) is a common progressive neurodegenerative disease, but treatment is not entirely satisfactory. At present, one of the main difficulties in the treatment of PD is the poor penetration of drug BBB. The researchers designed and selected the cell-penetrating peptide TP10 as the drug carrier, using a "click" reaction to conjugate dopamine (DA). The experimental results showed that the peptide drug conjugate had better pharmacokinetic and pharmacodynamic properties than DA. It can cross the BBB and enter brain tissue, has a low sensitivity to the O-methylation reaction of catechol-O-methyltransferase, or even lower than DA, and has a higher affinity for dopamine receptor 1 (D1) and dopamine receptor 2 (D2) receptors, as in the case of D1 receptors, much higher than DA. In drug-induced preclinical animal models of PD, anti-Parkinsonian activity is more pronounced compared to levodopa (L-DOPA). Therefore, the combination of drugs for the treatment of PD with CPPs may become a new strategy for the treatment of PD.

M6G-Ang

The analgesic effect of M6G after intracerebral injection is 50 times higher than that of morphine. However, the brain penetration of M6G is significantly lower than that of morphine, thus affecting its efficacy. Therefore, the M6G-Ang peptide drug conjugate, i.e., 1 Angiopep-2 peptide coupled to 3 M6G by disulfide bonds, was designed. Experiments have proved that intravenous or subcutaneous injection of An2-M6G peptide conjugate drug increases BBB permeability compared with M6G and morphine monomers, and it has greater and longer-lasting analgesic activity than the same dose of morphine or M6G, and An2-M6G shows the advantage of reducing constipation side effects. These results suggest that the use of brain-targeted peptide carriers as a novel BBB-permeable technology can be applied to the treatment of CNS diseases.

Methods for Improving Brain-Targeted PDC

Despite the many advantages of brain-targeted peptide conjugates, there are some limitations. Due to its lower molecular weight than biological macromolecules, poor stability, rapid renal clearance, and short half-life and cycle time, it may lead to limited drug efficacy. In addition, because there are a variety of enzymes in the gastrointestinal tract, which will degrade peptides in the gastrointestinal tract, such drugs are not suitable for oral administration, and the commonly used delivery method is intravenous injection, resulting in poor patient compliance. Currently, researchers are also using different techniques to overcome these challenges.

Modification of the Structure of Peptide

At present, researchers often use peptidomimetics, staple peptides, bicycle strategy, disulfide bond formation, and replacing L-amino acids with D-amino acids to enhance the stability of polypeptides and prolong the half-life. RAP12 peptide is derived from the miniaturization of receptor associated protein (RAP) and has binding affinity for LRP-1. However, when it is detached from the stable protein environment, RAP12 exhibits a weak α-helix fragment. Considering that the α-helix structure is a common structural motif for the peptide to mediate ligand-receptor interactions, the stapled peptide stapled RAP12 (ST-RAP12) was synthesized using peptide stapler technology. Compared with RAP12, the optimized ST-RAP12 has been shown to have higher α-helix content, binding affinity with LRP-1, and serum stability. In addition, ST-RAP12 exhibited enhanced internalization of bEnd.3 cells, U87 glioma cells, and human umbilical vein endothelial cells (HUVECs). In addition, ST-RAP12's ability to overcome BBB and blood–brain tumor barrier (BBTB) in vitro is also enhanced. ST-RAP12 peptide was further applied to modify the polymer material to construct ST-RAP12 micelles. The experimental results showed that the micelles could effectively penetrate BBB/BBTB and target gliomas in vitro and in vivo. In addition, ST-RAP12 micelles can effectively deliver PTX to glioma, prolong the survival time of glioma-bearing mice, inhibit tumor angiogenesis, induce glioma cell apoptosis, and have obvious anti-glioma effects. BT1718, which is in clinical trials, is obtained by conjugation of bicyclic peptide to mertansine (DM1) through a cleavable disulfide bond. Bicyclic peptide can specifically bind to membrane-type 1 matrix metalloproteinase (MT1-MMP), which is overexpressed in malignant tumors such as breast, lung, ovarian, and colon cancers. Compared with ADCs, BT1718 has a low molecular weight, is well distributed, can quickly penetrate and "kill" tumor cells, and has a therapeutic effect on advanced solid tumors. Numerous studies have demonstrated that Angiopep-2-modified nanocarriers significantly enhance brain distribution. A reverse isoform of Angiopep-2, named DAngiopep-2, was constructed to develop a brain-targeted drug delivery system. Although in vitro experiments showed that DAngiopep-2 had lower uptake efficiency in brain capillary endothelial cells compared to LAngiopep-2, it exhibited greater stability and achieved higher distribution in both normal brain tissue and intracranial glioblastoma cells when used in modified micelles.

Table.3 Peptide modification services at Creative Peptides.

Modification of Chemical Macromolecules

The charge of the polypeptide is also related to the drug clearance. Negatively charged peptide sequences have a longer half-life than positively charged peptides because the presence of anionic charges on the glomerular lining limits the filtration of anionic compounds in the urine. In addition, it can be done by increasing the size of the peptide and plasma protein binding to prevent the conjugate from being filtered out through the kidneys. One strategy is to conjugate polyethylene glycol (PEG) to brain-targeted peptide drugs, which can extend the half-life. The intrinsic properties of PEG make it an ideal candidate for modification: cheap, hydrophilic, biocompatible, non-immunogenic. It is one of the most widely used unnatural polymers for increasing the solubility of peptides, decreasing immune responses, and increasing the bioavailability of peptides. Each oxygen atom in the PEG polymer molecule can bind two to three water molecules, which greatly increases the mass and solubility of the compounds attached to it. The FDA has also approved a variety of pegylated proteins, such as PEG-bovine adenosine deaminase and PEG-α-interferon. However, PEGylated peptides with a molecular weight of at least 30 kDa will cause their vacuolation in various organs, such as kidneys, liver, spleen, bone marrow, etc. As a result, natural alternatives to PEG have been developed. The most prominent alternatives are PASylation and XTEN. XTEN is a genetically fused peptide composed of non-repetitive random fragments of six chemically stable amino acids: alanine (A), glutamic acid (E), glycine (G), proline (P), serine (S) and threonine (T). These amino acids are selected based on the principle of avoiding the possibility of affecting protein solubility, activity, or stability. Exenatide conjugation to XTEN significantly improves the pharmacokinetics of the peptide, extending its half-life in rats, mice, or monkeys by 65, 71, or 125 times, respectively. PASylation refers to polymers of proline (P), alanine (A), and serine (S). Polymers composed of these amino acids are thought to have a similar effect on the hydrodynamic volume of peptides as PEGs and are biodegradable, and have been successfully applied to more than 10 first-generation biologics, including human growth hormone, leptin, erythropoietin, exenatide, uricase, and coagulation factors, among others.

Strategies for Dosage Form Optimization

The physiology of the gastrointestinal tract poses challenges to the clinical application of most oral protein formulations. The stomach contains harsh acids and enzymes, making it essential to protect both the drug and its delivery system from degradation. Various strategies have been developed to enhance the oral bioavailability of peptide drugs, including acid-resistant coatings, intestinal enzyme inhibitors, mucus-penetrating peptides, and osmotic enhancers. Research has shown that anionic nanoparticles smaller than 100 nM can function as physicochemical osmotic enhancers to facilitate the oral delivery of proteins. Notably, these nanoparticles do not traverse the intestinal epithelium as transport carriers. instead, they bind to receptors on the intestinal surface, mediating the opening of tight junctions. Specifically, they interact with integrins and activate myosin light-chain kinase (MLCK) to temporarily increase intestinal permeability. This effect is reversible and does not cause tissue necrosis or inflammation. Additionally, acid-stabilized coatings have been employed by incorporating pH-sensitive intestinal polymers that remain intact in acidic environments and dissolve upon exposure to the more neutral pH of the intestine, triggering the release of the encapsulated drug.

Summary

Due to the existence of BBB, it is very challenging to discover and develop new drugs that are effective for the treatment of various CNS diseases. As a non-invasive drug delivery system, brain-targeted peptide drug conjugates can bring drugs into the brain parenchyma through BBB. Brain-targeted peptide conjugates with low molecular weight may have high permeability, efficient cell trafficking, low immunogenicity, and easier synthesis and purification compared to ADCs. This makes it another research hotspot in the field of targeted drug delivery after ADC. The factors influencing the brain-targeted peptide drug conjugate to become an effective drug for the treatment of CNS diseases are: (1) the efficiency of drug delivery to the brain. (2) the distribution of drugs in the brain. (3) the release of effective drugs and the accumulation in the brain. (4) the selection of linkers related to drug release and in vivo stability. (5) Receptor saturation, etc. Therefore, the selection and design of various components of brain-targeted peptide drug conjugates are particularly important.

However, due to the characteristics of peptides, their application is also limited. At present, researchers have also invented many new technologies to overcome these shortcomings, such as structural modification, conjugation with biological macromolecules, and dosage form modification. With the development of related technologies, such as novel screening platforms from peptide libraries of bacteriophages, yeast displays, neurotropic viruses, and stem cells, as well as target-specific in silico design, researchers will develop brain-targeted peptides with better BBB selectivity, higher transport capacity, and stronger metabolic stability. It is believed that in the future, the brain-targeted peptide-conjugated drug delivery system can make substantial progress and play its unique advantages in the treatment of CNS diseases.  

Reference

1. Woods, Ben, et al., Bioconjugate supramolecular Pd²⁺ metallacages penetrate the blood brain barrier in vitro and in vivo. Bioconjugate Chemistry 32.7 (2021): 1399-1408.