January 27, 2023

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Introduction of non-clinical safety assessment of peptide drugs

Introduction of non-clinical safety assessment of peptide drugs


Introduction of non-clinical safety assessment of peptide drugs.


This article is mainly based on the journal article “Mitra et al. (2020) Regulatory Toxicology and Pharmacology, 117 Discussing the development of therapeutic peptide products from the perspective of non-clinical safety assessment” and “Ypsilanti (2018) Health Sciences Eastern Michigan University, about synthetic peptide clinical The regulatory issues of the previous test are compiled.


Introduction of non-clinical safety assessment of peptide drugs



Peptides are a unique class of drugs and an important treatment method in global medicine. Since the world’s first peptide insulin was discovered in 1920, subsequent products include hormone replacements (insulin and corticotropin), antibiotics isolated from natural sources (bacitracin, gramicidin D and polymyxin B) ) Was developed one after another.


Since the 1970s, synthetic hormone analogs (Sarahine, desmopressin, and gonadotropin) have become the main focus of the therapeutic peptide industry.

Today, recombinant technology has been used to manufacture hormone replacements (parathyroid hormone). Hormones and glucagon) and analogs (duraglutide, insulin analogs and Teduglutide) can be used in many therapeutic areas. As of 2021, more than 100 peptides have been approved for use in humans.

Generally, polypeptides are chemically synthesized, derived from cells and derived from biotechnology, or isolated from natural products, and then added lipid conjugates to recombinant products or through chemical modification of natural derived products.


Peptide drugs cover many different types of peptide molecules and are manufactured in a variety of ways, including non-peptide modifications.

Although the exact definition of peptide drugs is a controversial issue, peptide-based therapies can range from 3 to 100 amino acids (AA) in length.


Peptide drugs are between small molecules and proteins in structure, and have the unique advantages of high specificity and low toxicity.

In terms of identifying new receptors and targets related to disease pathogenesis, a unique and valuable complete peptide library combined with high-throughput screening (HTS) technology has been successfully developed.

This technology has found signs of peptide screening. The compound is an important step in the treatment of diseases.


However, the inherent properties of certain polypeptides, such as metabolic instability and the need for parenteral administration routes, are major obstacles to the development and formulation of polypeptide drugs.

The above obstacles can be overcome by chemically synthesized peptides by using non-proteogenic amino acids to improve metabolic stability, or by using chemicals (such as polyethylene glycol, PEG) to enhance transport across the membrane.


In addition, the most challenging stage of peptide development is the preclinical stage. Since peptides cannot reach cell targets by diffusion like small molecules, nor can they directly bind to intracellular receptors after passing through cell membranes, it is extremely challenging to determine their pharmacology and pharmacokinetics through in vitro and in vivo tests.


Like any other biotechnology, the development and safety assessment of peptides also face various complex challenges. Including the lack of regulatory regulations in the development of peptide therapy has caused several problems.

These issues include, but are not limited to-regulatory regulations that need to be followed when developing chemically synthesized and biotechnology-derived peptides, strategies for toxicological laboratory animal model selection, whether the evaluation of genotoxicity and immunogenicity has added value, and whether it is necessary to Non-protein amino acid peptides were subjected to additional toxicological studies.


Because of the lack of specific medical registration regulations for peptide development in the world, the non-clinical safety assessment of such drugs can only rely mainly on the integration of existing small molecule drugs [International Coordination Union ICH S1-S5, S7-S8, M3 (R2 )] and the biopharmaceutical [ICH S6 (R1)] medical registration regulatory guidance regulations, but the guidance regulations for peptides are still limited.


The previous US Food and Drug Administration (US FDA) registration regulations stated that all protein and peptide products produced by cell culture, except antibiotics and hormone products, should be reviewed as biological agents.

The FDA CDER department reviewed the vast majority of peptides in the current data set. The only exceptions are Fc or albumin fusions (abirutide, dulaglutide, romiplostim), acalentide, the new 60AA recombinant kallikrein inhibitor and parathyroid hormone (84 AA) because they It is considered a hormone product.


The FDA changed the classification of peptide drugs in March 2020.

At that time, the Biologics Price Competition and Innovation (BPCI) Act officially announced that recombinant molecules larger than 40AA, regardless of their pharmacology, would be regarded as protein biologics.

Regardless of the production method, all peptides less than 40 AA will follow the New Drug Application (NDA) approach.


The summary basis of approval (SBA) analysis of therapeutic peptide products in the United States from 1998 to 2019 in this report includes a total of 47 peptides, 49% of which were chemically synthesized and 40% of them were biologically synthesized. Technologically synthesized, 13% is semi-synthetic, and 1% is obtained from natural sources.


In order to understand the medical registration requirements of therapeutic peptides, based on the analysis of the approval summary of therapeutic peptides, we found the research and development model, the medical registration regulatory regulations followed, and the strategies for the selection of toxicological experimental animal models, including: genetic toxicity and immunity Requirements for originality evaluation, evaluation of impurities, metabolites and safety pharmacology, safety and safety evaluation of peptides containing non-protein amino acids (NPAA). Its non-clinical toxicology development strategies are as follows:



General Toxicology

According to the wording of the current ICH guidelines, peptides derived from chemical synthesis and biotechnology appear to belong to ICH S6 (R1) regulations.

However, it is clear from the approved therapeutic peptide products that peptide drugs are always developed using small molecule drug methods similar to ICHM3 (R2).

This includes general toxicity studies in rodent and non-rodent species, non-clinical toxicology studies with high doses based on the maximum tolerated dose (MTD) setting (including later evaluation of genotoxicity, developmental toxicity, and reproductive toxicity), rather than considering Potency, binding affinity, target binding and/or receptor occupancy (RO).


Although the ICH M3 (R2) Medical Registration Guidance Regulation clearly states that therapeutic peptide products are outside the scope of this regulation, the ICH S6 (R1) guidance regulation recommends the development of chemically synthesized or recombinant peptide drugs under the principles of this regulation.

However, no matter what method of peptide synthesis is used for the approved therapeutic peptide products, most of them follow the ICH M3 (R2) guidelines and regulations for all peptides in rodents (rats) and non-rodents (dogs or cynomolgus monkeys).

Routine toxicology studies have been conducted in, and the highest dose explored and provided in the study is based on the maximum tolerated dose or limit dose.


Generally, the duration of chronic toxicology studies follows the guidance of ICH S4. Rodent studies usually last for ≥6 months, and the same non-rodent studies also last for ≥9 months.

For acute or shorter clinical dosing regimens, chronic toxicology studies with shorter durations can be used .




Developmental and Reproductive Toxicology (DART)

Since therapeutic peptide drugs can be used as hormone analogs or antagonists, and hormones are necessary for reproductive development and fetal development in many cases, developmental and reproductive toxicology studies are considered as safety assessments for therapeutic peptide drugs It is important.

Therefore, for all therapeutic peptide drugs, Seg I rodent reproductive toxicology studies, Seg II rodents and rabbit embryo-fetal development reproductive toxicology studies, and Seg III rodents prenatal and postnatal development reproductive toxicity studies should be carried out. 


In addition, reproductive toxicology also conducted larval toxicity studies on therapeutic peptide drugs. According to ICH S6 (R1) registration guidance regulations, biopharmaceuticals require major reproductive toxicology studies in non-human primates (NHP) animals.

However, in the process of obtaining approval for the registration of recombinant peptide drugs, it was discovered that in the reproductive toxicology experiment, the medical registration regulatory agency has not yet discussed the selection of experimental animal models related to peptide drug pharmacology.




Polypeptide drugs contain non-protein amino acids

Non-protein amino acids can be naturally occurring (such as sarcosine, ornithine or D-alanine) or non-naturally occurring (such as 1-naphthylalanine, 4-chloro-D-phenylalanine or Benzyl tyrosine). In polypeptide medicine, non-protein amino acids are usually added to the polypeptide in order to improve the binding affinity or stability of the polypeptide to the target.


Among the 47 peptide drugs approved after 1998, 45% contained at least one non-protein amino acid, 17% of which contained at least one non-natural non-protein amino acid, and most of these peptide preparations contained more than one Non-protein amino acids.


In theory, once the polypeptide is metabolized, it can release non-protein amino acids, which can then be incorporated into endogenous proteins in the body.

When non-protein amino acids are present in the structure of peptide therapeutics, even if there is no precedent for the use of these non-protein amino acids, the medical registration regulatory agency does not require other toxicological studies.

The only exception is that non-protein amino acids are also present as impurities in the bulk drug or drug product.


Therefore, for the vast majority of non-protein amino acids (natural or unnatural), even in the case of previously approved peptide drugs that have never used non-protein amino acids, it is extremely rare for independent non-protein amino acids to be specifically genetic or general toxic. Science research.


Among the two approved peptide drugs, the medical registration regulatory agency requires that the sponsor’s bivaluridine peptide drug (bivaluridine) must be subjected to acute and repeated dose toxicity studies in rats, because there are two non-protein amino acid raw materials. (DS) exists in the form of impurities.


For icatibant peptide drugs (icatibant), when impurities are found in two non-protein amino acid APIs, the medical registration regulatory agency requires the sponsor to conduct single-dose in vivo toxicology studies and genetic toxicology studies (bacterial reverse mutation Try, Ames test).





Immunogenicity (ADA) assessment

Chemically synthesized and recombinant peptide therapeutics, like proteins, have the potential to induce human immunogenic responses.

If peptide drugs are based on AA sequences that are not present in any endogenous protein, the production of anti-drug antibodies (ADA) will result in loss of efficacy and altered clearance. In most cases, this will mean increasing the dose level or switching to other drugs.


However, when peptide drugs are analogs of endogenous proteins or have significant overlap with the sequences of endogenous proteins, the production of ADA antibodies may mean antibodies against such endogenous proteins. If this problem exists, it will be A major safety issue.


Among the peptide drugs approved after 1998, 67% of the peptide drugs have ADA evaluated in non-clinical or clinical studies. Among peptide drugs evaluated for immunogenicity, 62% and 87% of ADAs were positive in non-clinical and clinical studies, respectively, but the sensitivity of non-clinical ADA tests in predicting clinical ADA positive results was only 72%.


In the past decade, the sensitivity of ADA determination and the method of measuring ADA in the presence of excess drugs in the serum have improved.

Therefore, almost every peptide drug tested after 2007 has a positive ADA response. The amino acid length of peptide drugs that produce ADA ranges from 8 to 84.


In addition, in clinically ADA-positive peptide drugs, most (66%) do not contain non-protein amino acids.

This indicates that neither the amino acid length of the peptide drug nor the presence of non-protein amino acids have a significant impact on its immunogenicity.

Among the drugs that tested positive for ADA clinically, 90% of the cases did not affect the pharmacokinetics (PK) of the drug or its efficacy.


Although the correlation of the ADA test with clinical response in non-clinical species has not been established, in all cases where the drug shows positive non-clinical ADA, it will most likely be ADA positive in the clinic.

Among the drugs that are not clinically evaluated for ADA in the clinic, 72% of the anti-drug antibodies are positive, while 87% of the ADA drugs in the clinic are positive, indicating that peptide drugs have a high tendency to produce antibodies.


However, in 90% of cases where ADA positive reactions have been observed clinically, no effect on PK or efficacy has been observed.

Even in the two cases where ADA affects the pharmacodynamic readings, it only accounts for the patients in clinical trials.

For a small part of the participants, this does not seem to be a major safety hazard.

However, it is still recommended to use relevant and sensitive detection methods to monitor the formation of ADA in the development of peptide drugs.




Impurity identification

According to the existing medical registration supervision and guidance regulations, the quality inspection standards of peptide drugs are based on the guidance regulations of small molecule drugs [ICH Q3A (R2) and ICH Q3B (R2)], or in accordance with the biopharmaceutical guidance regulations (ICH Q6B) related APIs and The standards for impurities and degradation products in drugs have not yet been determined.


The peptide drug sponsor conducted an independent identification study of impurities and degradation products in 57% of the peptides produced by chemical and semi-synthetic production.

These include in vitro genotoxicity tests and general toxicity studies using “spiked” or degraded batches.


For icatiban peptide drugs, the sponsor is required to conduct a three-month impurity identification study based on the identification and identification threshold of peptide impurities, instead of the traditional threshold mentioned in the ICH impurity identification guidelines.

Therefore, it is suggested that the future peptide recombinant drugs may require degradation product identification research.

For example, for the insulin Digolus and Detmir insulin, a one-month rat degradation qualification study was conducted for this type of peptide drug.


Because of the lack of clear guidance regulations on peptide drug impurities, and ICH Q3A (R2) regulations specifically indicate that peptides are not within the jurisdiction.

Although the small molecule impurities associated with the peptide drug process may be present in synthetic or semi-synthetic peptides, these impurities can usually be easily controlled by modern manufacturing techniques.


In contrast, the impurities related to the peptides produced in the peptide synthesis process are more of a problem.

Because they are similar in structure to the drug itself, and compared with traditional small molecules, the larger and relatively complex peptide molecular structure, so these are related to peptides. The impurities are difficult to detect, quantify and control.


Recombinant peptides can use methods similar to the ICH Q6B guidelines to treat these impurities as product heterogeneity, but chemically synthesized peptides are likely to contain similar heterogeneity, while still retaining the traditional impurity guidelines that focus on small molecules.

In this case, a more detailed and flexible approach is needed to deal with the impurity problem of peptide drugs.




Biotransformation and pharmacokinetic evaluation

Detailed biotransformation studies have been conducted on all chemically synthesized and semi-synthetic peptide drugs.

These studies include studies on the metabolism and activity of peptide drugs in vitro, structural characterization of metabolites, and quantitative studies on metabolites in metabolic kinetics and toxicology studies in vivo.

Among them, only a few recombinant peptide drugs used insulin glargine and insulin glargine radiolabeled peptides for non-clinical in vivo metabolite analysis.


In addition, the levels of these human polypeptide metabolites are lower than those in non-clinical laboratory animals, so no studies on the safety of polypeptide-independent metabolites have been conducted.

In addition, ICH S6 (R1) regulations specifically point out that biotechnology-derived drugs will be degraded into small peptides and a single AA.

Therefore, their metabolic pathways are fully understood, so it is considered that their biotransformation research is not necessary.


With one exception, if the major metabolites of the polypeptide will affect the pharmacological response, it is recommended to perform a quantitative analysis of the major metabolites in non-clinical and clinical studies to understand its impact on the pharmacology of the drug.




Genotoxicity assessment

According to ICH S6 (R1) regulations, recombinant products do not need to undergo genotoxicity assessment, because recombinant products will not directly interact with DNA or other chromosomal materials.

However, in peptide drugs approved after 1998, a complete standard genotoxicity assessment (including in vitro bacterial Ames and in vivo and in vitro chromosomal aberration tests) has been carried out for all chemically synthesized, semi-synthetic derived and natural peptide drugs.


Therefore, the reasons for the genotoxicity assessment of recombinant peptide drugs are unclear. Among the approved peptide drugs, no matter what method is used to synthesize peptide drugs, most of the peptides (including recombinant peptides) have been evaluated for genotoxicity, and the final result is judged to have no genotoxicity.


Another possibility is that when using the Ames analysis method to evaluate chemically synthesized or semi-synthetic peptide drugs, the impurities generated during the peptide synthesis process may cause potential genetic toxicological reactions.




Phototoxicity assessment

The phototoxicity assessment of peptide drugs is to use the 3T3 neutral red absorption test to evaluate the phototoxic potential of peptides in vitro.

However, the European Medical Association experiments pointed out that peptide drugs have some tendency to absorb ultraviolet light (usually a peak at 280 nm and a shoulder peak at 290 nm).


Because the aromatic amino acids in polypeptides can act as chromophores. This has nothing to do with any phototoxicity, and there is usually no need for photosafety testing of peptide drugs. In addition, according to ICH S10 regulations, peptide drugs are not covered by the regulations.

Therefore, it is not recommended to evaluate the phototoxicity of peptide drug therapeutics.




Safety Pharmacology

The ICH S6 (R1) and ICH S7A&B regulations provide specific guidelines on biological agents and small molecule drugs.

However, in the evaluation of safety pharmacological tests of peptide drugs, there is no specific regulation that peptide drugs must be tested for hERG inhibition and a comprehensive QT assessment.


Among the peptide drugs approved after 1998, 49% of the peptide drugs submitted in the NDA were found to perform hERG analysis and evaluation.

After the ICH S7B regulation was issued in 2005, 69% of the peptide drugs submitted for approval contained specific hERG analysis. It is currently not recommended to perform hERG analysis on peptide drugs submitted under a biological license application (BLA).


Interestingly, 67% of peptide drugs submitted by BLA include hERG analysis in their data packets.

In cardiovascular safety pharmacology studies, whether peptide drugs submitted in the form of NDA or BLA, 40% of the approved peptide drugs submitted are cardiovascular safety pharmacological studies conducted using rats, dogs or non-human primates.


However, less than half of peptide drugs are evaluated in comprehensive clinical QT studies, and it is not clear what criteria will be used to include or omit these studies.

Among the peptide drugs approved so far, no peptide drug with a definite QT interval extension has been found.



Carcinogenicity assessment

According to ICH S1 regulations, carcinogenicity assessment requires peptide drug testing in two non-clinical rodent models. 15% and 35% of approved peptide drugs were used to conduct carcinogenicity studies in one and two rodents respectively.

Since all peptide drugs are not genetically toxic, it is observed that about 50% of the carcinogenicity may be rodents The pharmacologically exaggerated results of the model.

Since peptide drugs are inherently non-genotoxic, it is recommended to test the pharmacological activity of peptides in rodents before conducting carcinogenicity studies, and to communicate fully with the medical registration and supervision unit.


In short, this freehand report aims to introduce the opinions of medical registration supervision units related to non-clinical safety testing of peptide drugs, and emphasize the main differences in the supervision of peptide drugs, and provide the toxicology, safety pharmacology and immunogens required for their development. The expectations of sexual assessment.


There is great ambiguity in the development of peptide therapeutics.

The analysis in this report provides a general framework for the non-clinical development of peptide therapeutics, and highlights the need for medical registration and regulatory agencies to address the genetic toxicity, impurities, and metabolism of peptide therapeutics. Establish special regulations for product and non-protein amino acid evaluation.


Another purpose of this report is to analyze the non-clinical toxicological development examples of approved peptide drugs, and provide information to Chinese peptide pharmaceutical companies to register therapeutic peptide products in the United States, with a view to understanding the requirements for supporting their peptide drugs Research and strategy development registered in the United States.






Introduction of non-clinical safety assessment of peptide drugs

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