Designing Early-stage Formulation Strategies for Anti-microbial Peptides (Amps): A Major Prerequisite for Combating Anti-microbial Resistance

Introduction:

At present, the world is witnessing an exponential increase in antimicrobial resistance (AMR) making it important for us to search alternatives to antibiotics in order to combat multi-drug resistant (MDR) bacterial infections. According to a study, drug-resistant infections were responsible for 1.27 million deaths in 2019, globally. The number of predicted deaths due to antimicrobial resistant infections is expected to surpass the number of deaths due to cancer by the year 2050, raising an alarming situation for all. In this context, several new antibacterial strategies are being tested, out of which, the use of antimicrobial peptides (AMPs) against MDR bacteria has been shown to give better hope in terms of a long-lasting solution to the problem.

AMPs are small peptides (10-50 amino acids long) that are released by a wide repertoire of organisms as a part of their innate immune response. Since these peptides are naturally produced, they are shown to be less toxic as compared to other antimicrobial agents. In fact, they have also been shown to possess broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria. The fact that AMPs have antibiofilm properties as well makes them more potential candidates as a probable solution to overcome the increasing problem of antibiotic resistance. However, despite these advantages, there are numerous limitations that need to be overcome in order to enhance the bioavailability and in-vivo activity of these peptides, so as to achieve a better therapeutic index. The low success rate with respect to AMPs being used as antibacterial agents is due to the fact that these peptides show properties like low chemical stability, considerable hemolytic effect, susceptibility to proteases, and tendency to lose bactericidal activity in presence of salts and serum. In this regard, various novel strategies are being considered, such as those involving the use of liposomes, lipids, nanoparticles, and micelles for formulation of the AMP-based drug.

Biologic drug formulation or pharmaceutical formulation is the process of reaching the final medicinal product from the active drug ingredient. This formulation is usually reached after various trials that include mixing of components in order to attain the desired stability, solubility, pH, and particle size.  The four major steps to be followed during the process of drug formulation are: determining the mode of action of APIs (active pharmaceutical ingredients) i.e. AMPs, finding the supporting ingredients that are biologically inert, identifying the interactions between the above substances, and following a manufacturing procedure that does not sacrifice the effectiveness of the API. Further, the formulation needs to be given in different dosage forms and doses, for example the concentration of drug to be given in the form of a tablet, might differ from the concentration used for the intravenous injection of the same drug due to the difference in absorbability, stability and the rate at which it is metabolized. Thus, drug formulation plays an important role in delivering the final form of drug into the patient’s body with as less side-effects as possible.

One of the recent drug formulation strategies is the use of nanocarriers. In scenarios where the AMP is unable to cross the cell membrane, nanocarriers can be used. These nanoscale carriers can be of different types, such as polymeric nanoparticles, metallic nanoparticles, liposomes, nanorods, and many more. Nanocarriers are also being used for targeted drug delivery. These can be made to open up and release the drug at certain defined pH. Nanoparticle-mediated delivery of AMPs and use of polymeric AMP carriers have also shown to give promising results. Another important formulation strategy is PEGylation where polyethylene glycol (PEG) is used to enhance drug solubility and biocompatibility. PEG is known to have ‘stealth’ properties that help improve the therapeutic potential of drug nanocarriers by escaping detection and destruction within the body, and thereby increasing its circulation. In one of the studies, the addition of PEG has shown to reduce the net charge or the zeta potential on the surface of liposome nanocarriers, thereby aiding in crossing the biological membrane. Besides, AMPs can also be modified covalently to alter parameters, such as absorption rate, permeability, stability, and biocompatibility. These covalent modifications are usually made in the amino acid side-chains so as to leave the main structure of the peptide undisturbed. There are various other formulation strategies such as those to reduce toxicity. In this context, an antimicrobial peptide, Cyclosporin A (CsA), released by Tolypocladium inflatum can be seen as a good example of a formulated peptide, used in the treatment of psoriasis and arthritis. When used for long-term treatment, CsA can become toxic and lead to several side-effects. In order to avoid this, lipids and surfactants are being used which mask the toxic effects of CsA, and make it more biocompatible. There are other drug formulation strategies that are employed based on the nature of the AMPs. For example, lipophilic substances can easily interact with the hydrophobic moieties of CsA. Polymeric lipidic substances are able to combine easily with these peptides and are responsible for stabilizing the system. In fact, in the presence of phospholipids, these peptides can form smaller and homogeneous crystals to increase solubility.

Another reason why drug formulation needs to be taken care of is because its metabolism might vary with its route of administration. There are substances that can be used to prevent the degradation of AMPs in the GI (gastro-intestinal) tract as well. The lipidic and polymeric carriers can be helpful for administering the AMPs orally, thereby shielding them from enzymatic degradation in the GI tract. These carriers can encapsulate the AMPs which slowly degrade after absorption, thereby providing a controlled and sustained release of the peptide. Designing of amphiphilic block copolymers and hydrophobic modification in the AMPs can be done to increase their permeability across the blood-brain-barrier (BBB). In case of lungs, although the pulmonary membrane is quite permeable to peptides, their entry may be restricted due to the presence of proteases. To escape degradation by proteases, lipidic and polymeric formulations of AMPs can be of great help.  Various liposomal encapsulations are being tested in this regard. For example, a liposomal formulation for polymyxin B sulfate has been shown to increase its activity against Pseudomonas aeruginosa strains. Nanoparticles have also been shown to be of help, especially in sustained drug release in the lungs.

Although drug formulation is already established as an important step, the importance of drug formulation in early-stages of drug-development process remains neglected. When developing a new therapeutic agent, its chemistry, manufacturing, and control (CMC) practices must involve not only a systematic substance characterization in order to understand the structure, its material properties and stability issues but must also address these properties early as a part of phase-appropriate biologic formulation strategy.

Once we have an idea of what all modifications we might need to make to our peptide in order to make it a reliable drug, we can plan our workflow accordingly and the drug can be tested likewise. Also, it is very crucial to ensure the safety of the drug in the very beginning and determining that it is non-toxic to our body, so that we are not left with such big loopholes at the end. Another very important reason why biological formulation of drugs needs a preliminary determination is because we also need to test the biocompatibility and toxicity levels of the inactive substances added to the drug formulation simultaneously with the active ingredient during our drug-development procedure. This early-stage determination can not only help in better analysis of the supporting ingredients suitable for our drug but also save a lot of time. Since these tasks are usually left to later stages, it not only becomes difficult to analyze the behavior of the drug but also forces us to perform multiple tests again. In this regard, reaching one of the finest end-product in a shorter time-period can be accomplished using a risk-based machine learning strategy where the identification of high-risk areas helps in initial filtering process and optimization. For this, all we need is huge amount of training data containing the in vitro measurements and properties of API molecules based on which we can design algorithms to predict successful drug-delivery processes to support the API activity.  Early-stage drug development is also crucial because it can be timely ensured in the early stage that the formulation has the potential to be manufactured at large-scale. Thus, if we pay attention to the biologic formulation from the very beginning of any drug designing process, things can get much easier in terms of saving time and futile efforts. One can now understand the reason for highlighting the importance of early-stage biologic formulation strategies.

One of the biggest reasons why early-stage drug formulation of AMPs is very important is that we have only a limited number of options available to cure infections caused by antibiotic-resistant bacteria. There are only a handful of antibiotics that are considerably effective against multi-drug resistant bacterial infections. Not only this, the number of APIs known to act as alternatives to antibiotics is even more limited. This is the reason why we need to try various different formulations for a single API (AMPs), in order to maximize the output. In this regard, early-stage biological drug formulation strategies for AMPs can help us move in the right direction to come up with effective AMPs to overcome antibiotic-resistance in bacteria.