Abstract: Oxidation is the second most common degradation pathway for

pharmaceuticals, after
hydrolysis. However, in contrast to hydrolysis, oxidation is mechanistically more
complex and
produces a wider range of degradation products; oxidation is thus harder to control.
The propensity
of a drug towards oxidation is established during forced degradation studies.
However, a more
realistic insight into degradation in the solid state can be achieved with accelerated
studies of mixtures
of drugs and excipients, as the excipients are the most common sources of impurities
that have the
potential to initiate oxidation of a solid drug product. Based on the results of these
studies, critical
parameters can be identified and appropriate measures can be taken to avoid the
problems that
oxidation poses to the quality of a drug product. This article reviews the most
common types
of oxidation mechanisms, possible sources of reactive oxygen species, and how to
minimize the
oxidation of a solid drug product based on a well-planned accelerated study.

1. Introduction
Safety and efficacy are key aspects of drug research and development, therefore,
formulations must be designed in a way that ensures appropriate bioavailability of a
drug and its physico-chemical stability over the determined shelf-life [1]. The
chemical
stability of a drug is an intrinsic property that is determined by its chemical structure.
The dosage form can lead to drug instability because of the presence of other
compounds
(e.g., excipients). Also, the drug manufacturing process, packaging, and storage
must be
monitored for drug stability. In this context, the problem of drug product stability is
an
very important area in drug research and development, not only for new drugs, but
also
for generic drugs

he chemical substance but not its chemical composition, which means that no
chemical
bonds are broken or formed [4]. The physical instability of a drug manifests as
changes
in its appearance, the release of the drug, polymorphic changes, adsorption, and
many
others [5]. On the other hand, chemical changes refer to changes in the chemical
structure
that arise from degradation of the drug substance and the reactions between the drug
and
the excipients in the formulation. These changes can reduce the drug potency, which
raises
efficacy concerns, and, at the same time, they can pose a safety risk, as the
degradation
products might be toxic
risk assessment for potentially mutagenic compounds. The ICH M7 (assessment and
control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential
carcinogenic risk) guideline is written on this topic [9], which describes, in detail,
the
process of toxicity assessment (the use of two complementary in silico systems, for
e.g.,
DEREK and Leadscope) [10,11], setting limits (threshold of toxicological concern
(TTC) or
median toxic dose (TD50) [12,13]), and analytical control (developing analytical
methods).
With the information from the forced degradation study, researchers can evaluate
the
stability of a drug under different conditions according to the amounts of degradation
impurities that are formed. After the structures of the degradation products have been
determined, there is the need to determine what types of chemical reactions are
involved
in the degradation of the drug (Figure 1). This information helps to predict the
reactions
that can occur with excipients and their contaminants

For drugs that are susceptible to oxidation, compatibility with some excipients might
become a problem. With the knowledge of the degradation products, a stability-
indicating
method can be developed, which is the starting point for the development of the final
methods for the analysis of a formulation [15]. Drug formulations are continuously
an alyzed during their development, with a specific focus on the stability of the
drug. This
information, in turn, influences the selection of suitable excipients until adequate
stability
of the formulation is obtained, e.g., the addition of antioxidants for oxidation-
susceptible
compounds. Even after the approval of a drug product, the suppliers of the excipients
might change during its life cycle or different qualities of excipients might be used
[16]. All
of these changes should be closely monitored from the analytical point of view, as
certain
degradation impurities might increase.

2. Oxidation Reactions
The following primary oxidative degradation mechanisms are known:
• Autoxidation (radical mediated);
• Nucleophilic/electrophilic (peroxide mediated);
•Oxidation that is mediated by single electron to dioxygen.
The term autoxidation classifies the oxidation of a substrate by molecular oxygen,
3O2. Autoxidation can start a chain process when the oxidized substrate generates
a
reactive species that subsequently attacks additional substrate molecules. This
mechanism
is also known as a radical chain reaction, where the addition of oxygen gives rise
to
hydroperoxides and their associated peroxy radicals (ROO•
) [22,23]. The radical chain
reaction is referred to as the Bolland–Gee mechanism [24].
At this point, we note that according to the recommendations in this review, we do
not
use the term free radical. In the context of physical organic chemistry, it appears
desirable
to cease using the adjective ‘free’ in the general name of this type of chemical species
and
molecular entities, so that the term ‘free radical’ might in the future be restricted to
those
radicals that do not form parts of radical pairs [25].
Chain processes consist of three concurrent reactions: initiation, propagation, and
termination [26] (Figure 3). Depending on the radical concentrations and the
specific
rate constants, each of these reactions will dominate within a particular time
domain,
which leads to the three distinct phases. The initiation step requires a radical
initiator that
generates radicals [27]. The initiation reaction can be triggered by hydroperoxides
(ROOH),
which are common excipient impurities
The initiation of oxidation reactions can involve the abstraction of H-atoms from
various moieties of the drug substance by the impurity-derived radicals. These can
result
from the reaction between hydroperoxides and trace amounts of iron or copper
ions,
whereby the result of both of these reactions is the same: the drug radical (D•
), here, copper,
is about 50 times more reactive than iron. This is followed by the reaction between
oxygen
from the air and D•
, which produces drug-derived peroxy radicals (DOO•
), which can
abstract hydrogen from another drug molecule. This is the key reaction in
autoxidation,
and once it takes place, the chain reaction is set. The chain can be relatively long,
where only
one initial radical is needed to produce hundreds of hydroperoxide molecules.
Reactions
that are involving peroxy radicals are relatively slow, so this is the limiting step in
the chain,
and peroxy radicals might start to accumulate. The hydroperoxide (DOOH) that is
formed
is the first stable oxidation product that can be identified
Penams are a class of β-lactams possessing an azabicyclo[3.2.0]heptane ring
system, a carboxylic acid moiety at C3, and a sulfur atom at position one (Figure 1).
The prototypic and most well-known penam is penicillin G (PNG or
benzylpenicillin), isolated from Penicillium chrysogenum (Fleming, 2001). The
other commonly used natural product penam is penicillin V, or
phenoxymethylpenicillin. In the absence of β-lactamases, the natural penicillins
show good Gram-positive activity but fail to inhibit the growth of many Gram-
negative organisms (Libby and Holmberg, 1945; Eagle, 1946; Eagle and Technical
Assistance of Arlyne D. Musselman, 1947; Nell and Hill, 1947; Thomsen and
Larsen, 1962). A great deal of creative synthetic chemistry has been directed to the
penam platform to modulate the spectrum of this class, predominantly via
modifications at the C6 acylamino group (R1 side chain). Clinically useful penam
derivatives have geminal dimethyl groups at C2, except tazobactam which is used
in combination therapies as a β-lactamase inhibitor. The penam amoxicillin is one
of the most often prescribed antibiotics in the United States, many other countries,
and in veterinary medicine (Tao et al., 2019).
Mechanism of Activity
β-lactam antibiotics act through structural mimicry of the D-alanine-D-alanine
motif in peptidoglycans of the bacterial cell wall (Figure 2). They inhibit bacterial
transpeptidases which catalyze the cross-linkage of peptidoglycan through the
formation of isopeptide bonds (Goffin and Ghuysen, 1998; Macheboeuf et al., 2006;
Sauvage et al., 2008). Peptidoglycan is an essential component of the bacterial cell
wall and plays major roles in protecting bacteria from environmental stress, ensuring
osmotic stability. Cell wall degradation and resynthesis are critical to bacterial cell
growth and division. The major target of β-lactams are PBPs, which are essential
peptide cross-linking enzymes and are essential for peptidoglycan synthesis. In the
presence of a β-lactam, the serine nucleophile attacks the lactam carbonyl, resulting
in a stable acyl-enzyme complex (Yocum et al., 1979; Waxman et al., 1980). This
action interrupts the integrity of the bacterial cell wall, diminishing the capacity for
growth and division and removing protection from osmotic or tensile stress. A
second major mechanism of action, specific to carbapenem β-lactams are the L,D-
transpeptidases. For example, faropenem efficacy against mycobacteria is due to
L,D transpeptidase covalent adduct formation (Lohans et al., 2019; Lu et al., 2020).

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