Talking about: Understanding the gene toxicity of drugs from the pharmacology & toxicology & warning structure

In recent years, the issue of gene toxicity has been one of the important content of drug development and even post-marketing drug quality evaluation, and it has even become the "flash point" and "burning point" in the industry! However, when most colleagues talk about the issue of gene toxicity, most of them only stay at the warning structure and the concept of 1.

01

What is genetic toxicity?

Genetic toxicity can be divided into four types: DNA damage, gene mutation, chromosome structure change and chromosome number change.

02

History of Genetic Toxicology

In 1927, genetic toxicology originated from HJ Muller's pioneering work.

In 1938, based on Muller's research, Sax discovered that X-rays can induce chromosomal structural aberrations in tradescantia pollen particles.

In 1946, Auerbach and his colleagues reported that nitrogen mustard can induce mutations in Drosophila.

By the 1970s, two events made it possible to use mutagenic data to assess risk.

In the same period, the second development changed the field of genetic toxicology, that is, Ames (1975) and his colleagues established a simple and cheap mutation test with Salmonella typhi; this test can detect chemical-induced Back mutation of the histidine locus, and can be combined with a foreign aid S9 metabolic system.

The Ames test has been widely used today, and further is the same as the in vivo micronucleus test in the 1970s.

Then, in 1994, Gatehouse et al.

Today, ICH, OECD and other institutions have formulated and promulgated a number of guidelines for genetic toxicology research, and they are constantly updating.

03

Guiding Principles issued in my country

my country issued the "Technical Guidelines for the Study of Drug Genotoxicity" on October 23, 2007.

In March 2018, in order to guide and standardize the research on drug genotoxicity, the State Food and Drug Administration revised the "Technical Guidelines for Drug Genetic Toxicity Research" and at the same time announced the "Drug Genetic Toxicity Research Guidelines" issued by the former State Food and Drug Administration in 2007.

Figure 2: Guiding principles issued by my country, source: http://samr.

Standard test set

The guiding principle points out: According to the genetic end point of the test, the test methods can be divided into three categories, namely gene mutation, chromosome aberration, and DNA damage; according to the test system, it can be divided into in vivo tests and in vitro tests.

Table 1: Domestic Guiding Principles and Standard Test Combinations

04

Guidelines issued by ICH

In 1995 and 1997, ICH respectively promulgated the genotoxicity guidelines S2A (specific guidelines for drug genotoxicity testing) and S2B (genotoxicity: drug genotoxicity test standard combination); the ICH genotoxicity steering committee was launched in June 2006 The revision of the genotoxicity guidelines has been held in the United States, the European Union, Japan and other places to discuss the revision work.

Figure 3: S2 (R1) guidelines issued by ICH

Standard test set

The guiding principle states: Choose one with more historical application experience, partly because this combination is recommended by the S2A and S2B guiding principles.

Table 2: ICH S2 (R1) Guiding Principles Standard Test Combination

05

Representative events of drug genotoxicity in history

In June 2007, Roche Pharmaceuticals launched the HIV protease inhibitor nelfinavir mesylate in the European market.
Because a classic genotoxic impurity, ethyl methanesulfonate, was found in its products, the EMA suspended it.
All markets in Europe.
The introduction of ethyl methanesulfonate is due to the fact that when the production equipment is cleaned, the ethanol is not completely dried and remains, and it reacts with the methanesulfonic acid in the bulk drug of nelfinafil mesylate to form ethyl methanesulfonate.

After Roche is required to completely solve the pollution problem, it still needs to supplement toxicity study data to better assess the risk of the genotoxic impurities of nerfinafil mesylate to patients.
It was not until Roche completely solved these problems that EMA resumed the relevant authorization of Nephenafil mesylate in the European market.
After the Nephenafil Mesylate incident, drug regulatory agencies around the world have put forward clearer regulations and requirements for genotoxic impurities.
Therefore, domestic and foreign pharmaceutical companies are increasingly engaged in the research and development of new drugs.
Pay attention to the control and detection of genotoxic impurities.

06

Common genotoxicity (impurity) problems in small molecule drugs

So, how to recognize and control genetic toxic impurities?

The structure of genotoxic impurities is diverse.
For most impurities, there are often insufficient toxicity or carcinogenic research data, so it is difficult to classify them.
In the absence of safety data support, most regulations and guidelines use the "warning structure" as a sign to distinguish between ordinary impurities and genotoxic impurities.
For impurities containing warning structures, (Q)SAR prediction and in vitro and in vivo genetic toxicity and carcinogenicity studies should be carried out, or the impurity level should be controlled below the threshold of toxicological concern (TTC).

For specific details on the warning results of genetic impurities, please refer to the warning structure "Development of structural alerts for the in vivo micro nucleus assay inrodents" issued by the European Union, or enter The Carcinogenic Potency Database (CPDB), which contains a list of thousands of carcinogens.
, Structural formula, CAS number, site of action, TTC value and a series of information.

ICH’s first guidelines for the study of impurities in chemical raw materials Q3A (R2) and Q3B (R2) for the study of impurities in preparations were firstly given by ICH.
For the first time in these guidelines, "for potential impurities that can produce strong pharmacological activity or toxicity, Even if its content is less than 0.
1%, further structural identification must be carried out.
" In the revised version, it is further clarified that "we must pay attention to potential genotoxic impurities in APIs" and "for very toxic genotoxic impurities, lower detection limits need to be formulated based on actual conditions", but it did not It puts forward a clear explanation for genotoxic impurities and their research and control issues.
At the same time, there are no specific requirements for its research principles, limit requirements and control strategies.

The EMEA Committee on Medicines for Human Use (CHMP) launched the "Guidelines for the Limits of Genotoxic Impurities", introducing the acceptable risk intake, that is, the concept of threshold of toxicological concern (TTC).
The limit value TTC (1.
5 μg/day) is set, which is equivalent to 1.
5 μg of genotoxic impurities per person per day, which is considered to be an acceptable risk for most drugs (the risk of cancer in life is less than 100,000 points one).
According to this threshold, the acceptable impurity level in the active drug is calculated according to the expected daily intake.
It is worth noting that TTC is only a probabilistic method and carries certain risks.
If there is a genotoxic impurity whose toxicity is unknown, assuming that its daily intake is within the TTC range, the probability of it causing cancer will be below 10-5, so the limit value (TTC) does not mean that there is no risk at all.
PS: There are different thresholds and algorithms for different types of drugs.

Table 3: Classification of genotoxic impurities in drugs

07

M7 guiding principle ~ very important

M7 was adopted by the ICH Steering Committee in the 4th stage of the ICH process in June 2014; in the 4th stage of the ICH process, the final draft was recommended for adoption by regulatory agencies in the European Union, Japan and the United States.

M7 aims to provide a feasible framework for the identification, classification, definition and control of mutagenic impurities to limit the potential carcinogenic risks.
At the same time, it is intended to supplement ICH Q3A (R2), Q3B (R2) and M3 (R2) to support clinical trials and non-clinical safety studies of drugs on the market.
PS: M7 is not applicable to APIs and preparations for advanced cancer indications defined in the scope of ICH S9.

M7 again gives the concepts and data of TTC, TD50, 1.
5μg/day, etc.
, and emphasizes the problem of genotoxic impurities after drug changes after marketing; see the table below.

Table 4: The genotoxic impurity problems faced by drug changes after marketing

08

Conclusion

The most fundamental issue of genotoxicity is safety, and whether a substance is safe and whether there is genotoxicity depends on experiments and data to prove it.
So far, we have known a lot of gene toxic substances and how to judge the warning structure, but there are still NDMA incidents! Regardless of the cause of the incident, the result is that the quality of the medicine has gone wrong! However, when quality problems arise, as a pharmaceutical practitioner, more reflections should be where the problem lies? How should we make "safe, effective, and quality controllable" drugs!

In summary, it is the content of genetic toxicity that the author understands at the moment.

reference:

1.
ICH Tripartite Coordination Guidelines.

2.
Guiding Principles S2 (R1) of Human Drug Genetic Toxicity Tests and Results Analysis.

3.
ICH.
S2(R1): Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use.
2011

4.
ICH.
M3 (R2): Non-clinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals.
2009

5.
FDA.
Guidance for industry and review staff: Recommended approaches to integration of genetic toxicology study results.
2006

6.
OECD.
Guideline for testing of chemicals No.
471: Bacterial reverse mutation test.
1997

7.
OECD.
Guideline for testing of chemicals No.
473: InVitro mammalian chromosomal aberration test.
2016

8.
OECD.
Guideline for testing of chemicals No.
488：Transgenicrodent somatic and germ cell gene mutation assays.
2013

9.
OECD.
Guideline for testing of chemicals No.
489：Invivo mammalian alkaline comet assay.
2016

10.
OECD.
Guideline for testing of chemicals No.
490：Invitro mammalian cell gene mutation tests using the thymidine kinase gene.
2016

11.
M7 DNA （）.

12.
M7(R1)：DNA（）.

13.
.
CNKI

14.
http://samr.
cfda.
gov.
cn/WS01/CL0087/226424.
html

15.
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