This document provides an overview of non-clinical studies conducted for a new drug called QMU1221 to treat transient liver disease in adult women. It finds that while some preliminary studies like toxicity testing were conducted on rats and dogs, many crucial non-clinical studies required by regulators were not fully carried out due to a tight budget. This includes insufficient general toxicology, reproductive toxicology, and ADME studies. Further long-term testing is needed in multiple species to fully characterize the drug's toxicity profile, support later phases of clinical trials, and satisfy regulatory expectations.

1. PHAR2202 Drug Design And Analytical Methods
Answer:
Introduction
The procedure of developing a drug involves two processes: clinical and non-clinical
studies. Clinical studies are performed on people and are aimed at examining a behavioral,
medical, or a surgical intervention. It is the primary technique used by researchers to test
the safety of a new drug, diet, treatment, or medical device. Basically, the study helps to
know the effectiveness of the drug and if its side effects are less harmful than the existing
drugs. On the other hand non-clinical studies refer to studies conducted on animals and
cells but not humans. The purpose of the test is to examine the safety of the new substance
being studied. The non-clinical testing investigates any harmful impact of the substances on
a body caused by the pharmacology of the new substance, including genetics, toxicity, and
carcinogenicity, amongst others. The information from non-clinical studies aids in clinical
studies planning, in which the information is used in deciding the starting dose and range of
doses in the clinical studies. Similarly, it suggests the clinical signs to be looked for to
identify any harmful effects (Sacaan et al., 2020).
Various protocols are used when conducting non-clinical trials (Sarkar et al., 2019). The
protocols usually follow GLP (Good Laboratory Practice) regulations. In the initial
preclinical process, also referred to as Go/No-Go decision, the substance under test must go
through various steps, like pharmacokinetics (to determine availability of drug) and ADME
(absorption, distribution, metabolism, and elimination) as well pilot studies whose
objective is to investigate the safety of the candidate drug including general toxicology,
mutagenicity, safety pharmacology, and genotoxicity (Mustonen et al., 2017). The
preliminary studies are not subject to mandatory compliance with the regulations of GLP.
The studies aim is to investigate the safety of the candidate drug to acquire the first-hand
data about its admissibility in various systems appropriate for advance deliberations.
Nonetheless, there are other compulsory studies that need to be conducted with respect to
the GLP standards due to safety exposure to human beings. The mandatory tests include
safety pharmacology, genotoxicity, and dose toxicity, and should be performed prior to the
process of IND (Investigational New Drug). The non-clinical studies set usually must cover
information required for safe switch of medicine to humans from test animals, basically on

2. the basis of NOAEL (Non-Observed Adverse Effect Level) acquired from the research of
general toxicity (Iansante et al., 2021). Following IND approval, are other GLP experiments
used to evaluate chronic toxicity, genotoxicity, developmental and reproductive toxicity,
and carcinogenicity, which are carried out during the phase of clinical development (Ulrich
et al., 2018). Nevertheless, the need to perform the studies relies on the clinical application
purpose of the candidate drug.
In this report, a critical evaluation of non-clinical data for a new drug (QMU1221) developed
to treat transient liver disease in adult women, for a period of half a year (6 months) has
been done. Phase 1 studies were successfully conducted on healthy volunteers, and results
given. The next step is phase 2 studies that will involve patients and will go for a period of 3
months. The drug had been tested on rats and dogs for up to 4 weeks. Similarly, a 1-week
drf study was done in NHPs. In the rats, the target organs were the ovaries and testes.
Goals Of Study: Non-Clinical Testing Of Drugs
The goals of conducting non-clinical tests of drugs before rolling them out for human use
are, characterization of potential adverse effects of the drug, characterization of
pharmacokinetic profile, characterization of essential pharmacodynamic effects or proof of
principle, guidance towards safe application in clinical studies in humans by determining a
safe starting dose and providing monitoring guidelines for the study, and provision of
enough data for concluding that users/patients are not exposed to any risk during use
(Hoogendoorn, 2019; Wallis et al., 2018; Sacaan et al., 2020).
Phase 1 Studies
Usually, in phase 1 studies, the steps involved are toxicology, reproductive toxicology, and
ADME. In this study, all the above mandatory steps in phase 1 were conducted, albeit for the
minimum recommended period due to the tight budget.
Toxicity
The main target organ for the study was the liver. Consequently, it was imperative to know
the effect of the substance on the livers of the rats used in the study.
Reproductive Toxicology
For reproductive toxicology, the testes and ovaries were the reproductive organs picked for
analysis after the test.
ADME
The preliminary ADME studies were performed under the metabolite 1, metabolite 2, and

3. NOAEL for human, rat, dog, and NHP. The test results were recorded and are shown in the
results section. The NOAEL was done in mg/kg/day.
Results
Toxicity
In all the species tested, the liver was affected in the following ways
Increased enzymes in the liver
Increased weight
Increased hepatocyte hypertrophy
High doses led to cell neurosis
However, reversibility was not investigated
ADME
The early ADME studies results are shown below.
Preliminary QSAR Analysis
The preliminary QSAR analysis done for the parent and metabolite 1 yielded the following
results:
Mixed inconclusive results for genotoxicity was recorded, in spite of being negative for
bacterial reversion
The parent showed to possess much shorter half-life of elimination than the metabolite 1
Further, the metabolite 1 indicated ability to inhibit angiogenesis
Other Test Results
The other test data showed the following results;
The bacterial reversion (Ames) test turned negative
There was a positive result on in vitro in mouse lymphoma cells
The preliminary developmental drf indicated effects observed without maternal toxicity in
rats
Rabbits were not evaluated
No further studies were conducted
Questions To Ask About The Gaps/Incompleteness Of Information
Despite having gone through phase 1 so far, it appears most pre-clinical trials were left out
in the steps to fit the tight budget. Crucial steps of preclinical studies including GLP, IND,
NDA (new drug application), and ADME were seemingly not carried out properly
(Motoyama et al., 2018).

4. In addition, there is insufficient data concerning observation of both macroscopic and
microscopic observations.
Due to the tight budget, the execution of phase 1 was insufficiently done. Phase 1 of non-
clinical studies must involve both general toxicology and reproductive toxicology studies.
Similarly,
Reversibility in the effect of the drug on the liver was not investigated. Investigating the
reversibility was crucial for knowing whether the adverse effects on the liver were
reversible or permanent. If reversible, then include the remedy in the non-clinical study
test.
Why rabbits were not evaluated.
Apart From Studies Already Mentioned, What Else Is Needed To Satisfy Regulatory
Expectation?
The mandatory tests include safety pharmacology, genotoxicity, and dose toxicity, and must
be performed prior to the IND (Harris & Rhodes, 2018). The non-clinical studies package
usually must contain data required for safe switch of medicine to human beings from test
animals, basically on the basis of NOAEL acquired from the studies of general toxicity.
Following IND approval, are other GLP experiments used to evaluate chronic toxicity,
genotoxicity, developmental and reproductive toxicity, and carcinogenicity, which are
carried out during the phase of clinical development (Ventrella et al., 2019). Nevertheless,
the need to perform the studies relies on the clinical application purpose of the candidate
drug.
Species Recommended For Further Toxicity Testing
In the preliminary and phase 1 part of the non-clinical study, the drug was tested in rats and
dogs and the test lasted for 4 weeks. Also, a 2-week drf study was done in NHPs, albeit no to
GLP.
Further toxicity testing should be conducted on all the species to further study the toxicity
of the drug on the liver as well as the reversibility of the toxicity of the drug on the liver.
Also, further toxicity testing should be done on more rats to test for the effects seen in the
preliminary developmental dwarf in the absence of maternal tox.
In addition, since rabbits were not evaluated in the previous tests, they should be subjected
for further toxicity tests and evaluation to enrich the tests database and increase variety of
data.

5. Studies Needed To Support Phase 2
Phase 2 of the studies is conducted for the non-clinical studies during human clinical trials.
The studies needed to support phase are reproductive toxicology, chronic toxicology, and
carcinogenicity
Reproductive Toxicology
The reproductive toxicology tests are conducted in the clinical studies, together with
teratogenic potential evaluation. It is the most difficult test applied by the regulatory bodies
and it is a precondition for authorization of new drugs (Rahalkar et al., 2018). The
reproductive test is necessary because the drug is capable of affecting the reproductive
system and activities in the following ways:
Effect on fertility as well as initial development of embryo to fetus.
Effect on the final development of embryo to fetus (teratogenicity)
Effect on development of pre-birth and post-birth, including maternal function.
Chronic Toxicology
After given a green flag to commence clinical studies, other non-clinical toxicity tests such
as chronic and non-chronic studies should be carried out. The evaluation of toxicity is
usually classified according to a chronological scale, like the acute studies which are
conducted for verifying the effect of the medicine with the aid of single or repeated-dose for
a day. Normally the chronic studies are regarded as studies that consume more than 90
days (Lahn, 2020).
Carcinogenicity
Carcinogenicity studies are normally conducted in phases 2 and 3 on non-clinical studies
during human clinical development, with the help of a rodent species, more so rats.
Additionally, the study is recommended for performing other in vivo assays that are capable
of providing more data on the carcinogenic substance’s sensitivity, like a shortened
duration test on transgenic rat/mouse or a long duration carcinogenicity test in other
species of rodents (Paul, 2017). The long duration carcinogenicity study in rats is normally
carried out for a minimum of 2-year treatment period with 3 or 4 doses of the control and
the test article. Basically, in these non-clinical studies, the least dose is the highest dose
commended for human beings, whereas the maximal dose is the MTD acquired in the initial
safety tests (Fung, 2020).
The carcinogenicity test requires use of 50 to 80 animals per gender/group. Regulations
and rules of this test requires that the performance of the study to be done in harmony with
the principles of GLP, with spf (specific pathogenic-free) animals as well as the
histopathological analysis, in which in excess of 50 types of tissues are analyzed by a

6. professional veterinary pathologist who has a vast experience in carcinogenesis.
ADME (In Vivo And In Vitro)
The in vivo pharmacokinetics assay enables the quantifiable assessment of ADME of a new
drug. The information is significant as it helps in predicting the most suitable dosage and
the most desirable posology protocol to be applied. The in vitro tests are developed and
authenticated to enable safety assessment for discovering the probability of the substances
being toxic. Nonetheless, in most cases, these assays complement the in vivo tests. ADME is
carried out in phase 2 of the non-clinical studies as well as in other phases and preclinical
tests (de Almeida et al., 2020).
What Should Be Included In The New Studies For Investigating The Toxicity Profile
Identified So Far?
Preliminary Ames tests aid in evaluating a probable genotoxicity effect as well as to detect
genetic modifications and changes in organisms subjected to the drug. In this study,
preliminary Ames test was conducted but he results were not detailed. Therefore, in the
new studies the Ames tests should be intensively conducted to attain its full benefits.
Further toxicology tests should be included in the new studies to investigate the toxicity
profile. The additional safety tests include mutagenicity tests, local tolerance and safety
pharmacology, and acute toxicity (Martínez Muñoz, 2018). Similarly, reversibility should be
included in the new studies. Investigating the reversibility is crucial for toxicity profile
investigation.
Current Conclusion On The Toxicity Profile
In this critical evaluation of QMU1221, several safety steps and phases of non-clinical
studies have been reviewed. Within the review several classifications of toxicology (safety
steps) have been specifically looked at. The non-clinical safety steps include the general
toxicology that should be conducted in the first and second phase, sand lasts for 3 to 4
months. The second one is the reproductive toxicology which spans across phase 1, phase 2,
and phase 3 of non-clinical studies of human clinical trials. Carcinogenicity is the other non-
clinical safety step that is carried out in the phase 2 and phase 3 of non-clinical studies
during clinical human trials.
Studies That Will Be Needed To Support Phase 3
Phase 3 of the studies is conducted for the non-clinical studies during human clinical trials.
The studies needed to support phase 3 include reproductive toxicology and carcinogenicity.
Reproductive Toxicology

7. The reproductive toxicology tests are conducted in the clinical studies, together with
teratogenic potential evaluation. It is the most difficult test applied by the regulatory bodies
and it is a precondition for authorization of new medicine.
Carcinogenicity
Besides reproductive toxicology and general toxicology, the carcinogenicity study is always
needed for medicine proposed for constant treatment spanning 6 or more months. In these
instances, the carcinogenicity tests must be conducted prior to the market entry of the
substances, but never prior to the commencement of clinical tests (Paul, 2017). The
carcinogenic assay could be needed whenever there are substances known to belong to
known groups of carcinogens or when chronic toxicology shows indication of carcinogenic
probability, or when there is convincing indication that the substances and/or their
corresponding metabolites get retained in the organism for an extended duration.
ADME (In Vivo And In Vitro)
The in vivo pharmacokinetics assay enables the quantifiable assessment of ADME of a new
drug. The information is significant as it helps in predicting the most suitable dosage and
the most desirable posology protocol to be applied. The in vitro tests are made and certified
to enable safety assessment for discovering the probability of substances being toxic.
Nonetheless, in most cases, these assays complement the in vivo tests. ADME is carried out
in phase 3 of the non-clinical studies as well as other phases including the preclinical step.
References
de Almeida, A. A. C., de Oliveira Ferreira, J. R., de Carvalho, R. B. F., dos Santos Rizzo, M., da
Silva Lopes, L., Dittz, D., ... & Ferreira, P. M. P. (2020). Non-clinical toxicity of (+)-limonene
epoxide and its physio-pharmacological properties on neurological disorders. Naunyn-
Schmiedeberg's Archives of Pharmacology, 393(12), 2301-2314.
Fung, V. S. C. (2020). Integrating traditional Chinese (herbal) medicines into risk-based
regulation-With focus on non-clinical requirements to demonstrate safety. Journal of
Traditional Chinese Medical Sciences, 7(2), 88-94.
Harris, M., & Rhodes, T. (2018). Caring and curing: Considering the effects of hepatitis C
pharmaceuticalisation in relation to non-clinical treatment outcomes. International Journal
of Drug Policy, 60, 24-32.
Hoogendoorn, K. H. (2019). Advanced therapies: clinical, non-clinical and quality
considerations. In Pharmaceutical Biotechnology (pp. 357-402). Springer, Cham.
Iansante, V., Brooks, A., & Coney, L. (2021). Considerations in the Design of Non-Clinical

8. Development Programmes to Support Non-Viral Genetically Modified Mesenchymal Stromal
Cell Therapies. Pharmaceutics, 13(6), 823.
Lahn, M. (2020). The Development of a Drug: A Pharmaceutical Drug Development
Perspective. Phase I Oncology Drug Development, 1-15.
Martínez Muñoz, L. (2018). Non-Clinical Contribution to Clinical Trials during Lead
Optimization Phase. Behavioral Sciences, 8(1), 17.
Motoyama, S., Takeiri, A., Tanaka, K., Harada, A., Matsuzaki, K., Taketo, J., ... & Mishima, M.
(2018). Advantages of evaluating γH2AX induction in non-clinical drug development. Genes
and Environment, 40(1), 1-7.
Mustonen, E. K., Palomäki, T., & Pasanen, M. (2017). Oligonucleotide-based pharmaceuticals:
Non-clinical and clinical safety signals and non-clinical testing strategies. Regulatory
Toxicology and Pharmacology, 90, 328-341.
Paul, G. R. (2017). Defining dosimetry and implications for aerosol presentation for non-
clinical development of respiratory drugs (Doctoral dissertation, Cardiff University).
Rahalkar, H., Cetintas, H. C., & Salek, S. (2018). Quality, non-clinical and clinical
considerations for biosimilar monoclonal antibody development: EU, WHO, USA, Canada,
and BRICS-TM regulatory guidelines. Frontiers in pharmacology, 9, 1079.
Sacaan, A., Hashida, S. N., & Khan, N. K. (2020). Non-clinical combination toxicology studies:
strategy, examples and future perspective. The Journal of Toxicological Sciences, 45(7), 365-
371.
Sarkar, C., Jamaddar, S., Mollick, B., Akter, M., Ahmed, T., Mahir, M. I., & Islam, M. T. (2019).
Importance and the unique aspects of modalities for conducting non-clinical and pre-clinical
studies of compartmental and non-compartmental analysis. Pharmacologyonline, 3, 1-15.
Ulrich, P., Blaich, G., Baumann, A., Fagg, R., Hey, A., Kiessling, A., ... & Weir, L. (2018).
Biotherapeutics in non-clinical development: Strengthening the interface between safety,
pharmacokinetics-pharmacodynamics and manufacturing. Regulatory Toxicology and
Pharmacology, 94, 91-100.
Ventrella, D., Forni, M., Bacci, M.L. and Annaert, P., 2019. Non-clinical models to determine
drug passage into human breast milk. Current pharmaceutical design, 25(5), pp.534-548.
Wallis, R., Benson, C., Darpo, B., Gintant, G., Kanda, Y., Prasad, K., ... & Valentin, J. P. (2018).
CiPA challenges and opportunities from a non-clinical, clinical and regulatory perspectives.

9. An overview of the safety pharmacology scientific discussion. Journal of pharmacological
and toxicological methods, 93, 15-25.