The delivery and formulation of cyclic peptides also remains an important aspect in peptide therapeutics research and drug design, and plays a crucial role as a limiting step in the drug development process, of which the physiochemical limitations have been considered as the bottleneck in peptide drug research and development. For peptide therapeutics, this delivery challenge primarily includes metabolic degradation, low membrane permeability, and rapid clearance. Although these factors are slightly improved by the inherent stability to proteolysis of cyclic peptides, as well as the preorganization in the tertiary structure that the macrocyclization provides, the physiochemical properties of cyclic peptide therapeutics are still often unfavorable due to gastrointestinal degradation, limited absorption through epithelial barriers, and clearance via hepatic metabolism and renal filtration. In terms of formulation, this also closely follows the physiochemical properties of the small molecule as well, and it is critical to balance the target recognition through 3D structure with drug-like properties required for delivery.
Cyclic peptide therapeutics have very poor oral bioavailability due to the rapid degradation in the gastrointestinal (GI) tract as a result of their limited stability as well as enzymatic degradation, and formulation approaches had to be developed to combat this. However, they are more amenable to formulation than their linear counterparts for parenteral, oral, nasal and pulmonary administration. The decreased conformational entropy of cyclic peptides resulting from the conformational restriction of cyclic backbones can be beneficial in drug formulation. The structural rigidity provided by the cyclic backbone can confer metabolic stability on cyclic peptides, in part due to the resistance to proteolytic enzymes that recognize and cleave more flexible peptide substrates. Cyclic peptides may also exhibit improved membrane permeability as a result of the pre-organization of the molecule into a structure that can interact favorably with the membrane, an attribute that can be modulated by specific modifications. The inherent structural stability of cyclic peptides, as well as the lower conformational entropy that is a result of the constrained structure, not only confer advantages in terms of stability and membrane permeability but also provide a rich array of possibilities in terms of formulation. The limited conformational flexibility of cyclic peptides can be advantageous for the formulation of stable and homogeneous drug products. Additionally, the chameleonic behavior of cyclic peptides can be harnessed in formulation design to transiently modify the physicochemical properties of the drug to improve membrane permeation. The inherent predictability of such structure, which is afforded by the macrocyclic scaffold, allows the facile and predictable chemical modification of the peptide scaffold to improve the pharmacokinetic and pharmacodynamic properties. Application of advanced drug delivery systems and formulation approaches in the development of cyclic peptide drugs is key to optimizing the PK/PD properties of cyclic peptides for effective delivery of the drug to the site of action and desired therapeutic effect.
Delivery route selection for cyclic peptide drugs can have a significant impact on their bioavailability and therefore the choice of route is an important consideration. It also has implications for patient acceptability and compliance. For small molecule drugs, oral administration is the most convenient. For injectable drugs, the principal routes of delivery are subcutaneous, intramuscular and intravenous injection, which have varying degrees of acceptance by the patient and impact on their availability, and consequently also their therapeutic index. For each route, formulation can be important to address the challenges of delivery, and can involve the use of carriers to help solubility, stability and absorption. In recent years, the number of cyclic peptides being developed has increased, and this is driving interest in their delivery by alternative routes. Most peptides are administered parenterally to date. Formulations for such routes can be as simple as a buffered solution, or can involve more complex considerations, including various excipients or strategies that extend the duration of action (depot formulations). Oral administration of peptide drugs, which in the past was believed impossible, has now been developed for a number of cyclic peptides, which have better stability in the gastrointestinal tract. Other possible routes include intranasal and pulmonary, both of which avoid first pass metabolism through the liver.
Fig. 1 (A) Routes of nano-drug administration and (B) various barriers encountered by micellar systems before they reach the tumor site.1,5
Parenteral routes of cyclic peptide administration include intravenous (IV), subcutaneous (SC), and intramuscular (IM) administration. IV administration affords immediate and complete bioavailability and allows for tight control over drug exposure, making it the preferred delivery for acute, high-dose treatment indications. Intravenous injection does not encounter any barriers to absorption, but formulation must take care to ensure peptide solubility, long-term stability, and prevention of aggregation under conditions of the injection fluid and infusion times. Subcutaneous injections have the advantage of more sustained release over time, and are generally the preferred method of parenteral administration, making them an ideal drug delivery system for chronic administration. These formulations are typically in the form of viscous gels, but can also be microsphere-based or formulated with lipid suspensions, which act as depots for the drug. The resulting drug depot at the site of injection slowly releases peptide, which diffuses into the systemic circulation. These depots allow extended release of the peptide, and because they are generally injected at home, offer convenience, improved patient compliance, and overall better quality of life. However, they are also prone to injection site reactions, including erythema, pain, and lipodystrophy. Local inflammation caused by the peptide or formulation can cause some of these reactions. The cyclic peptide structure may contribute to this depot effect, as the macrocyclic structure protects against enzymatic degradation in subcutaneous tissue, causing the peptide to slowly dissolve and be released over a period of days to weeks. Because subcutaneous injection does not pass through the liver first, hepatic first-pass metabolism is also avoided, which can be an important consideration for peptide stability. IM administration can also be used, and represents an intermediate pharmacokinetic option between a traditional IV bolus and subcutaneous depot formulation. Intramuscular injection of peptide drugs is quickly absorbed from muscle tissue into the systemic circulation. Injection into muscle tissue carries some risk of nerve damage. Formulation for intramuscular injection often uses oils or viscous suspensions to control release. The injected peptide may be dissolved, or in the form of microcrystals that slowly dissolve after injection.
The oral route of administration is generally the most difficult for cyclic peptides due to enzymatic degradation in the gastrointestinal tract and membrane permeability limitation. A variety of approaches have been attempted to improve oral peptide bioavailability. This includes the co-formulation of peptides with permeation enhancers such as sodium N-(8-(2-hydroxybenzoyl)amino) caprylate (SNAC) that improves stability and intestinal membrane transport of peptides. Improved bioavailability and clinical activity have been reported for orally administered semaglutide and somatropin co-formulated with SNAC. Enteric coatings, enzyme inhibitors and mucoadhesive systems are also used to protect the peptides from degradation in the stomach and improve absorption in the intestine. Oral formulations of exenatide and insulin have been developed, and are in clinical evaluation.
Alternative routes of administration such as nasal, pulmonary, and transdermal delivery systems have also been applied for cyclic peptides. Nasal delivery is a non-invasive route of administration that can bypass the gastrointestinal system. Lipid-based nano-delivery systems have been studied for nasal delivery of cyclic peptides. Pulmonary delivery is another non-invasive route of administration using inhalation. This route is typically for local delivery to the lungs but has been explored for peptide delivery such as insulin. Transdermal delivery systems, such as patches and microneedles, can offer controlled release and reduced systemic exposure. This in turn can result in a reduction in adverse effects. Microneedles have also been used to improve absorption of insulin and other peptides through the outer skin layer. When chronic conditions that require continual management are present in patients, utilizing alternate methods of drug delivery can result in improved compliance and therapeutic outcomes.
Delivery vehicles (alternatively delivery systems or nanocarriers) for cyclic peptides are a collection of nano and micro technologies, specifically engineered to circumvent the physicochemical and pharmacokinetic shortcomings often associated with macrocyclic drugs. In particular, such formulations serve as protective "platforms" to prevent drug degradation, uptake and release mediators that can tune absorption rates, and matrices that are capable of targeted delivery and controlled release of a given cyclic peptide at specific sites of disease while maintaining drug stability, identity, and activity. While diverse in architecture and property, existing vehicles are most generally classified as lipid- or polymer-based, which typically confer different physical characteristics and which can be further modified to a given desired end goal (including therapeutic need, administration pathway, and target tissue). Lipid-based delivery vehicles take advantage of the biocompatible and biomimetic properties of naturally-derived phospholipids and other amphiphiles to deliver peptides encapsulated in either lipid bilayer vesicles or lipid matrices (often in a solid state) that can both protect the loaded drug from environmental degradation and promote interaction with biological membranes. Polymer-based drug carriers, on the other hand, make use of the chemical and mechanical stability, tunable biodegradability, and versatile chemistries of either synthetic or naturally-derived polymers to form peptide-encapsulating microspheres, hydrogel matrices, and polymer conjugates that can be further functionalized to allow for the addition of targeting moieties or ligands, and that can be designed to allow for sustained release. As such, the decision to pursue one vehicle type over another can be quite complex, and depend on a number of practical and clinical factors, including but not limited to: release time frame, target tissue, production scale-up and commercialization, and regulatory challenges. In recent years, therefore, many delivery approaches have involved a more hybrid system that combines both lipid and polymer elements to capitalize on both their unique advantages. Overall, then, modern delivery vehicles have evolved away from simply passively containing a drug, but are now being developed to also actively facilitate cellular uptake, avoid immune response, and other properties in response to disease conditions, thus enhancing and expanding the identity of cyclic peptide formulation from a solution to an active therapeutic.
Lipid-based delivery systems have been successfully employed for the delivery of cyclic peptides. Lipid-based nanostructures form versatile platforms that have been used for several different drug classes. These systems have been designed to take advantage of the amphiphilic properties of lipids and often result in stable and biocompatible formulations. Liposomes are lipid based vesicular structures that can be used for the delivery of multiple classes of drugs. Liposomes are composed of phospholipid bilayers and can entrap both hydrophilic and lipophilic drugs. Lipid nanoparticles (LNPs) including liposomes, solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) have been explored for the delivery of various cyclic peptides with often high loading capacity and enhanced stability. In fact, SLNs have been shown as a delivery system for the treatment of eye inflammation with cyclosporine A and for the treatment of psoriasis with calcipotriol. SLNs are composed of a solid lipid matrix and are often prepared by high-energy methods like homogenization and ultrasonication. Lipid-polymer hybrid nanoparticles have also been developed and are a combination of lipid and polymer based systems that can offer improved in vivo cellular delivery efficacy. These delivery systems can be functionalized for targeted delivery of therapeutics by modifying lipid composition and particle size. These systems can also be used to encapsulate a wide range of drug classes.
Fig. 2 Liposome-based co-delivery.2,5
Polymers, such as PLGA, have also been used commonly due to their biocompatibility and biodegradability. PLGA microspheres have been investigated for controlled release of drugs, and sustained release of a drug over a long period is possible. Hydrogels are 3D polymer networks, which can contain high amounts of water, and have the ability to allow localized and sustained release of drugs. They can be stimuli-responsive in design, and this can be used to create a controlled release of a drug in response to a stimulus such as a change in temperature or pH. PEG conjugates are cyclic peptides conjugated to polyethylene glycol (PEG) chains. This can result in an increase in solubility and decrease in immunogenicity, and may have improved pharmacokinetic properties. These systems can also be used to improve the stability of cyclic peptides, and increase the circulation time in the bloodstream. Polymer based systems continue to be a heavily researched area, with new approaches such as stimuli-responsive polymers and hybrid systems being investigated for targeted and controlled drug delivery.
Smart and targeted drug delivery systems aim to incorporate two features into therapeutic platforms. The first is responsiveness to a physiologic trigger, i.e., a feature that causes a drug to be released in response to a specific physiologic signal. The second feature is directivity, which aims to direct drug activity to the site of action, while avoiding healthy tissues. These strategies can solve one of the key problems with cyclic peptide therapeutic development, which is how to concentrate the drug activity at the site of action to a therapeutically meaningful level without off-target interactions and systemic toxicity. This is often a particular challenge for the high-potency macrocycles that tend to have wide distribution across tissues. While traditional drug delivery approaches have focused on bulk enhancements of peptide stability and circulation time, these often have broad distributions and lack the spatial selectivity needed for the more complex and inhomogeneous environments found in many diseases like cancer. Smart drug delivery systems solve this problem by either engineering materials that will respond to physicochemical changes in the body such as pH, enzymatic microenvironments, or temperature, or by engineering them to specifically bind tissue- or cell-specific receptors, to then drive uptake into the target cells. The application of these techniques to cyclic peptides converts the drug from a systemic agent to a targeted one, where drug release is coordinated in space and time with the disease state. In addition to providing a higher therapeutic index, this can allow for lower doses of drug, reducing both the cost of production and potentially reducing immunogenicity from chronic exposure at high levels. Targeting and stimuli-responsive elements can be combined in a single carrier to provide multi-modal drug delivery systems which first home in on the site of disease using receptor recognition, then release the drug in response to a microenvironmental stimulus. This type of system is especially relevant for cyclic peptides targeting intracellular pathways, where both escape from endosomes and cytosolic stability are desired, and must be coordinated with targeting and stimuli-responsive release. The stimuli-responsiveness of these systems requires careful characterization of sensitivity to specific stimuli, biocompatibility, and reproducibility in manufacturing, but has the potential to more closely align drug delivery with the biology of the disease state, and as such is an active area of research.
Stimuli-responsive delivery systems have the unique capability of turning on or off in response to specific signals or environmental changes. This is advantageous when compared to the previously mentioned methods because it can be turned on or off in physiological settings. This can be particularly useful in targeting the specific areas or cells which the cyclic peptide will benefit most. These systems can be tailored to respond to different stimuli, including pH, enzymes, and temperature changes, among others. pH-responsive systems are particularly useful for targeting tumor tissues, as they tend to have lower extracellular pH compared to healthy tissues. Carriers can be modified with pH-responsive polymers that change conformation or are cleaved at a specific pH to encapsulate the cyclic peptide and release the drug specifically at the tumor site. Enzyme-responsive systems take advantage of overexpressed enzymes at the disease site such as matrix metalloproteinases (MMP) at the tumor site or cathepsins at an inflamed site. The carrier can be modified with a peptide linker that is susceptible to the specific enzyme and cleave to control release. Temperature sensitive systems have phase transition or structural changes in response to different temperatures for localized drug delivery.
Targeted delivery systems can be used to direct cyclic peptide therapeutics to specific tissues or cells. One approach is receptor-mediated uptake, in which cyclic peptides are conjugated to a ligand that binds to a receptor that is overexpressed on target cells. For instance, folate-conjugated cyclic peptides can be used to target folate receptors that are commonly overexpressed on cancer cells. Another approach involves using antibody–peptide conjugates (APCs), which combine a monoclonal antibody with a cyclic peptide therapeutic. The antibody targets the APC to a specific antigen that is expressed on the target cell, allowing for precise delivery of the cyclic peptide. Targeted delivery systems can also be designed to take advantage of unique biological processes, such as the overexpression of specific receptors on inflamed tissues or the tumor microenvironment. By incorporating these targeting strategies with novel delivery vehicles, highly effective therapeutic agents can be developed that are tailored to specific diseases and patient populations.
Formulation is a big hurdle for cyclic peptides with regard to both storage stability and reconstitution. Peptides are sensitive to pH, temperature, and the nature of the excipients, and aggregation and degradation can be a problem, and have a major impact on the efficacy and safety of the drug candidate. Aggregation can be minimized with the incorporation of surfactants (such as polysorbates 80) in the formulation, which stabilizes the peptide and prevents aggregation during both the manufacturing process and storage. Excipients, also known as stabilizers, are also used in order to maintain the peptide in its native form, and also to prevent aggregation (common stabilizers include sugars and polyols). In order to convert a peptide solution into a dry powder, lyophilization (freeze-drying) can be used. Lyophilization can also be used to prevent aggregation. Packaging in moisture-resistant materials can also be important, especially with regard to long-term stability of a formulation, and protection against hygroscopicity. Aggregation and oxidation are two major challenges that can impact the stability and efficacy of cyclic peptides. Peptide aggregation can be influenced by peptide concentration, temperature, pH, and interactions with formulation excipients. Several strategies can be employed to minimize aggregation. Incorporation of surfactants in the formulation can help stabilize the peptide and prevent aggregation during storage. Stabilizers such as sugars and polyols are also used to help maintain the peptide in its native form, and prevent aggregation. The pH of the solution can also be adjusted to modulate the net charge of the peptide, thereby reducing interactions that could lead to aggregation. For oxidation prevention, antioxidants can be added to the formulation to help neutralize free radicals and prevent oxidative degradation. Proper storage conditions, such as maintaining the appropriate temperature and avoiding exposure to light, can also be important in preventing oxidation.
Emerging technologies in the delivery of cyclic peptides have the potential to improve their pharmacokinetics and therapeutic outcomes. One of these technologies is the development of oral peptide tablets. Oral semaglutide tablets are a recent example that has shown the potential for oral delivery of cyclic peptides. These tablets use advanced formulation strategies to improve peptide stability and absorption in the gastrointestinal tract. They often include permeation enhancers such as sodium N-(8-(2-hydroxybenzoyl)amino) caprylate (SNAC) to facilitate peptide uptake by the intestinal epithelium. The oral tablets have shown improved bioavailability and clinical efficacy, providing a more convenient administration route for patients. Enteric coatings that protect peptides from the acidic environment of the stomach and mucoadhesive systems that enhance peptide absorption in the intestines are also being developed as strategies for oral delivery of cyclic peptides. These emerging technologies are expanding the potential for oral administration of cyclic peptides, which may lead to improved patient compliance and therapeutic outcomes. Peptide–nanoparticle hybrid systems are also emerging as potential delivery systems for cyclic peptides. These hybrid systems combine the therapeutic potential of peptides with the favorable properties of nanoparticles. Lipid-based nanoparticles such as liposomes and lipid nanoparticles (LNPs) can encapsulate cyclic peptides, protecting them from enzymatic degradation and enhancing their stability. Polymer-based nanoparticles such as PLGA microspheres and hydrogels can provide controlled release profiles and can be engineered for targeted delivery. The hybrid systems can be further functionalized with targeting ligands to enhance tissue specificity and reduce off-target effects. These developments in peptide–nanoparticle hybrid systems are opening up new possibilities for the development of effective and versatile cyclic peptide therapies.
A major driver in the coming years for cyclic peptide delivery is the development of "smart" delivery systems that are ultimately personalized for the patient based on their individual disease signature, genetic profile, and even real-time physiological feedback. This is a critical step-change from formulating a drug with one or a few options that it may or may not match with a particular patient's disease microenvironment, to truly intelligent systems that can be personalized based on what is known about the patient prior to treatment and once they have initiated therapy. Smart drug delivery systems for peptides will be layered with multiple components that can be thought of as "intelligent" functions. This will include disease targeting moieties that recognize patient-specific tumor antigens or inflammatory proteins or other cell adhesion ligands to promote accumulation of the therapeutic peptide at the site where it is needed. Targeting may also be further combined with individualized stimuli-responsive release functions that respond to the conditions of the disease microenvironment, such as pH, enzymes, or redox, which can often be measured in an individual patient prior to treatment and matched to an equivalent stimulus-responsive carrier chemistry. Biosensors are also being developed that can be incorporated into the peptide delivery vehicles to facilitate closed-loop control of release in response to circulating biomarkers. These can be incorporated into implantable depots or injectable gels as well as oral drug formulations. Feedback control is particularly useful for cyclic peptides with narrow therapeutic windows, where maintaining the drug concentration in plasma in the optimal range is critical. Personalized delivery systems for peptides are also being designed for individual patients. These systems take into account individual patient factors like genetics, disease state, and metabolic profile, aiming to tailor the delivery of cyclic peptides to each patient's unique needs to improve treatment outcomes.
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