NL-PEPTIDES DELIVERY™ technology as an innovative method for creating new generation peptides

Synthagen Laboratories

NL-PEPTIDES DELIVERY™ technology as an innovative method of producing next-generation peptides A comparison of the action and durability of traditional peptides versus an innovative method of synthesis and oral delivery of peptides.

Summary: In practice, only a few traditional peptides find application due to their biological instability and rapid degradation. The solution to this problem is peptide modification, which allows stable and effective forms of peptides to be obtained. Next-generation peptides, produced through synthesis using NL-PEPTIDES technology, have been enclosed in a double-capsule form created within the NL-PEPTIDES DELIVERY™ system, enabling the maximization of peptide efficacy and stability.

List of abbreviations: • AFP – Pharmaceutically Active Peptides • NL-PEPTIDES – Next-Generation Peptides • NL-PEPTIDES DELIVERY – Oral Delivery Technology

Keywords: • peptide • peptide bond • analog • modification • salting-out • amidation • acetylation • synthesis • technology • salting-out • arginine

Introduction

NL-PEPTIDES DELIVERY™ technology, as an innovative method, allows a therapeutic effect to be achieved that surpasses the action of traditional peptides. The efficacy of NL-PEPTIDES DELIVERY™ has been confirmed based on responses to issues related to the production of peptides that reach the intestine intact and are subsequently absorbed in full. The innovative NL-PEPTIDES DELIVERY™ technology is protected by patent law as a new form of peptide analogs for the development and commercialization of oral peptides, combined with a specialized delivery system.

WHAT ARE TRADITIONAL PEPTIDES

From a purely chemical standpoint, peptides occur in an unbranched form and have only two specific ends. One of them is called the amino terminus, where an amino acid with a free α-amino group is present. The other is called the carboxyl terminus or C-terminus, where an amino acid with a free α-carboxyl group is present. Peptides are chemical compounds built, similarly to proteins, from amino acids. They are the subject of broad interest, fulfilling important biological functions. Many hormones and neurotransmitters are in fact peptides. In the case of endogenous peptides, they act antimicrobially, functioning as the body's defense system. Naturally occurring peptides are regarded as attractive compounds of therapeutic significance due to their high degree of activity, low toxicity, and lack of drug interactions. In medical practice, only a few peptides find application due to their biological instability and rapid degradation. The solution to the aforementioned problem of peptide stability is their synthesis, which allows stable peptide forms to be obtained. The same applies to the synthesis of peptides from natural sources, which are used, among other things, in the production of vaccines.

THE PEPTIDE BOND

Carbon, as a result of the reaction of the α-carboxyl group, binds to the nitrogen of the α-amino group via a single bond — a peptide bond. It is generally assumed that this bond is formed as two structures that remain in a defined mutual equilibrium. The C–N bond transitions to C=N and vice versa. Rotation about the C=N axis is not possible, which is why the peptide bond is sufficiently rigid to possess the characteristics of a double bond. Amino acids that participate in the formation of a peptide bond lose fragments of their molecules — specifically the –OH group from the carboxyl group and –H from the amino group. For this reason, amino acids found in peptides and proteins are called amino acid residues. The resulting peptide bonds are stable, and their breakdown can only occur under the action of strong bases and acids at simultaneously high temperatures.

PEPTIDE SYNTHESIS

Depending on the peptide we wish to obtain, an appropriate method of synthesis is required. In a brief explanation, we will attempt to present peptide synthesis in relation to its size. To obtain a dipeptide, a reagent must be used that leads to activation of the carboxyl group of the acylating amino acid, or the acylating amino acid must be converted into an anhydride. A more laborious and complex process is synthesis in the case of larger peptides, which are obtained from the dipeptide by removing the protecting group from the amino group of the N-terminal amino acid and acylating it with the next N-protected amino acid. This process is particularly time-consuming, because the aforementioned steps are repeated until the peptide with the planned sequence is obtained. In the case of obtaining large peptides, the best-performing and simplest method is the Merrifield method. This method is carried out in the solid phase. The C-terminal amino acid is attached to the polymer, where the next amino acid is then coupled, until the desired chain length is achieved.

PEPTIDE ANALOGS

In response to the unstable nature of traditional peptides, their analogs are designed and produced. Peptide analogs are defined as chemical compounds in which one atom is replaced by another relative to the parent compound. The general structure of the peptide remains unchanged. Peptide analogs include analogs with a helix structure as well as β-turn and β-sheet analogs. In the former, helices are one of the key structural elements of bioactive peptides. Stabilizing short oligomer fragments in a helical conformation results in increased activity. In β-turn and β-sheet analogs, D-amino acid residues or β,γ,δ-amino acid residues are inserted. Peptide analogs allow us to obtain new peptide compounds that are more stable, while simultaneously finding application across a broader range of indications and enabling innovative solutions to problems associated with the action of existing pre-analog forms.

PEPTIDE ANALOGS THROUGH MODIFICATION

Traditional peptides, despite their undeniable and numerous advantages, also have many limitations related to their application. The search for new, maximally stable peptide analogs with a broad spectrum of action is the result of the instability of traditional peptides. Analogs of traditional peptides containing the key sequence of the peptide are enriched with modifications carried out within the peptide chain or the side chain of amino acid residues present in the sequence. The introduction of amidation and acetylation contributes to improved metabolic stability and selectivity. Among the known examples of modifications, depending on the action profile that the peptide is intended to exhibit, are N-terminal acetylation of the peptide, cyclization, labeling with fluorophores, C-terminal amidation of the peptide, and D-amino acids.

THE PEPTIDE SALTING-OUT PROCESS

The salting-out process involves changing the charges on proteins. The protein charges are neutralized by the anions and cations of a salt. The protein molecules do not attract each other and do not form aggregates, and the protein itself is precipitated as a result of losing its hydration shell. The salting-out process is reversible. In the reverse process, the salt is removed by dialysis or its concentration is reduced by the addition of water. Salting-out, through the addition of an arginine molecule, leads to the formation of a stable form of the peptide and is an innovative method for ensuring peptide stability and, consequently, expanding the biological activity of peptides.

ACETYLATION AND AMIDATION OF A PEPTIDE

N-terminal acetylation of a peptide involves the attachment of acetyl radicals to substrates — which are compounds with an NH2, OH, or SH group — with the participation of the enzyme N-acetyltransferase. The source of the acetyl radical is acetyl-CoA. The primary function of N-acetyltransferases is to facilitate the joining of the acetyl group with the amino group of aromatic amines and hydrazines (the N-acetylation reaction), i.e. the detoxification of potentially toxic exogenous compounds. When peptide bonds are broken and, consequently, the polypeptide chain is fragmented, carbonyl groups are formed. The oxidation of a protein molecule by a hydroxyl radical begins with the removal of a hydrogen atom at the α carbon of the amino acid. The resulting alkyl radical reacts with oxygen to form an alkylperoxyl radical, which transitions into an alkyl hydroperoxide. The alkoxyl radical formed from it can be converted into an amino acid residue hydroxylated at the α carbon, or it can lead to fragmentation of the polypeptide chain. The presence of the alkoxyl radical promotes fragmentation of the polypeptide chain. Cleavage of the peptide bond can occur via α-amidation or diamidation. The N-terminal peptide formed during α-amide fragmentation contains an amide group at its C-terminus, while the second peptide contains an N-α-ketoacyl derivative at its N-terminus. Fragmentation via the diamide pathway is characterized by the formation of an N-terminal peptide containing a diamide structure and a peptide derived from the C-terminus of the protein molecule containing an isocyanate structure at its N-terminus.

WHAT ARE NL-PEPTIDES™

In response to the challenges surrounding the production of peptides that reach the intestine intact, we created a group of new peptides called NL-peptides, also referred to as next-generation peptides, designated by the abbreviation AFP. The characteristics of pharmaceutically active peptides reflect their superiority over traditional peptides. They are distinguished primarily by their extended longevity and resistance to changes in pH and temperature, both during product storage and in the gastrointestinal environment, where upon ingestion of the capsule and its transport to the small intestine, the peptide will not undergo degradation. The NL-peptide group is characterized by the ability to mimic naturally occurring proteins and by high metabolic stability, which means that NL-peptides, acting like natural proteins, are absorbed intact from the intestine and do not undergo degradation during absorption.

THE FORMATION OF NL-PEPTIDES™ THROUGH SYNTHESIS

The chemical description of NL-peptides produced through modification via synthesis is discussed below. The technology operating on the principle of salting-out the peptide with arginine has been enhanced through synthesis involving N-acetylation of the amino terminus of the peptide combined with simultaneous amidation of the carboxyl terminus of the bond. The totality of the modification processes carried out results in a 10-fold increase in the stability of NL-PEPTIDES and the aforementioned ability to mimic natural protein.

NL-PEPTIDES DELIVERY™ TECHNOLOGY

In undertaking the task of creating a modern technology for the oral delivery of peptides, it was essential to resolve issues concerning, first, the creation of a simple technology based on versatility with respect to a wide range of products, as well as the simultaneous production of several effective products at one time, through a method that enables the development of a system allowing for such a mode of operation. Existing problems related to absorption had been addressed through the use of the aforementioned peptide modifications alone. In our case, in addition to modifying the peptide, we focused on the undeniably important challenge of resolving the issue related to peptide transport from the moment of oral administration, through passage to the small intestine, up to the point of compound absorption. The solution to the aforementioned technological problems is the creation of a new, simple, and effective peptide delivery technology, which we refer to as NL-PEPTIDES DELIVERY™.

THE ACTION PROFILE OF NL-PEPTIDES DELIVERY™ TECHNOLOGY

The action profile, based on NL-PEPTIDES DELIVERY technology, primarily enables the peptide to reach the small intestine via a specially designed coating surrounding the NL-peptide. Beyond simply delivering the peptide to the small intestine, it is important that the entire local environment within the intestine is conducive to the absorption of the peptide delivered there. This issue was significant due to the presence of peptide-degrading enzymes — peptidases — in the intestine. The main enzymes that degrade peptides are proteolytic enzymes called proteases, which prevent the peptide from continuing its journey by degrading it in the small intestine. Another problematic factor, which we have resolved, is the poor absorption of the peptide itself through the wall of the small intestine.

ADVANTAGES OF NL-PEPTIDES DELIVERY™ TECHNOLOGY

Focusing on the specific action profile of NL-PEPTIDES DELIVERY™ technology, it is based on the development of a new double capsule with protective coatings, which resolves the issue concerning both the delivery of the peptide to the small intestine and its passage through the intestinal wall, as well as the degradation of the peptide at that location. The protective coating, forming the outermost surface of the capsule, shields the compound from pH changes and gastric acid, enabling the peptide to reach the intestine. Through the use of a protease inhibitor in the capsule composition, the peptide — which is otherwise susceptible to degradation — does not break down, while simultaneously lowering the local intestinal pH and preparing its environment for peptide absorption. An absorption enhancer has been used to improve the absorption of the peptide through the small intestine. The NL-peptide has been surrounded by a coating that separates it from the protease inhibitor. The inhibitor itself is an acid, which in combination with the peptide could degrade it. By separating it from the acid using a capsule-within-a-capsule approach, we have obtained a stable pharmaceutical composition that does not degrade during storage.

HOW NL-PEPTIDES DELIVERY™ TECHNOLOGY FUNCTIONS IN THE DIGESTIVE SYSTEM

Next-generation peptides are produced through synthesis using NL-PEPTIDES technology. Enclosed in the form of a double capsule created within the NL-PEPTIDES DELIVERY™ system, they follow a defined pathway through the digestive system upon oral administration. The swallowed capsule reaches the small intestine intact, where the protease inhibitor is released, thereby creating an appropriate absorption environment by lowering the intestinal pH to 5.5. In the subsequent stage, the active ingredient and absorption enhancer are released, allowing the peptide to be effectively absorbed by the body.

RESULTS OF USING NL-PEPTIDES DELIVERY™ TECHNOLOGY

A comparison of the results of oral delivery in the form of a double capsule versus nasal delivery of the peptide allows for the determination of the superiority and efficacy of one of the delivery methods. The concentration of the peptide upon oral delivery of our capsule is far greater than the concentration of the peptide upon nasal delivery. The same doses of peptide were used in both the capsule and the nasal spray during the study. The results of these studies clearly demonstrate the superiority of our technology over nasal delivery.

THE RESULT OF SYNTHAGEN LABORATORIES' WORK

The task we set ourselves concerned the creation of a new technology for increasing the efficacy of peptides. The NL-PEPTIDES DELIVERY oral peptide delivery technology is based on innovative peptide therapy combined with the novel oral delivery method we developed. A multi-channel presentation of our technology allows for a precise understanding of each of its stages.

Visually, the results of our work can be seen in the presentation.