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Preformulation in early drug development (small molecules), part 2

Updated: Dec 12, 2021

Welcome to My Blog series in preformulation for drug development

In this third series of preformulation blog I will talk about stability aspects of small molecules in early development. If you find that the reaction kinetics and mechanism is too dry, just read through; the reward will be worth it.

To stabilize a drug product, you need to know how a drug substance falls apart. You can do that only if you understand the laws that govern the degradation. In the next blog series, I am going to address strategies using formulation excipients (stay tuned and follow blogs). Biologics will follow the small molecule series.


Reaction (chemical) kinetics is the study of rates of chemical processes. Chemical kinetics includes investigations of how different experimental conditions can influence the speed of a chemical reaction and yield information about the way the compound degrades.

Reaction mechanism is the step-by-step sequence of elementary reactions by which chemical changes occur.

Energy of activation is the energy that must be overcome for a chemical reaction to occur (Svante Arrhenius). Transition state is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest energy along this reaction coordinate.

Reaction Kinetics

There are a few questions that need to be addressed. For example, how is reaction kinetics helpful in drug development; is this too academic? If you go too deep, it might become academic; however, you need to get the answers you need for your goal, i.e., stabilization of your drug product. To stabilize a drug product, we need to know how the API (active pharmaceutical ingredient) degrades and what is needed for a reaction to occur: collision (requires mobility; solution vs. solid-state), orientation/proximity, and energy of activation (e.g., heat). To get the answers, pharmaceutical scientists perform forced degradation. The first use of the forced degradation studies is for analytical method development. There is a saying that discovery starts with measurements. The better the measurements, the more reliable the data and the decision process are. Therefore, do not cut corners on forced degradation.

Another question is why do we need to know the pH-rate profile and why do we even need extreme pH’s like pH 1 and 13? One of the major degradation pathways for a pharmaceutical compound is hydrolysis and you will find different packaging barriers for humidity protection for the marketed drugs. Therefore, we need to know the pH of the maximum stability. But why lower and higher pH values? The answer is that your data helps the analytical development and can give feed-back to process chemists, where extreme pH’s are used either in sample preparation or in unit operations. In addition, a pH-rate profile that covers the whole pH range can better define the maximum stability (i.e., minimum degradation) and there are multiple cases where the profile is more complex (e.g., bell-shaped curve).

The first step in the study of a reaction always involves some consideration of mechanism of reaction (vide infra). At the most elementary level, the mechanism of a reaction will specify any intermediates involved in the overall reaction and involves the determination of the empirical rate law. The empirical rate law states how the rates of formation of products (disappearance of reactants) depend on the concentrations of reactants, concentration of products, and catalysts, while all other variables (e.g., temperature, pressure, ionic strengths) are held constant. The rate law for the elementary step is directly related to the stoichiometry. If the empirical rate law for some reactions does not correspond to the stoichiometry for the reaction, the reaction must involve more than one elementary step, and must involve at least one intermediate.

For reactions involving a single elementary step, there are simple first-order reactions (e.g., isomerization), second-order reactions, consecutive reactions, and parallel reactions (in this case, it is important to define the rate determining step). To simplify the process, the reaction kinetics (A + B ->C) is performed in pseudo-first order conditions, [B]>>[A]. A plot of ln([A]/[Ao]) vs. time gives a straight line and the slope of this line is the pseudo-first order rate constant. A plot of the pseudo-first order rate constant vs. pH gives the pH-rate profile and thus, the pH of maximum stability.

The Arrhenius equation gives the dependence of the rate constant k on the temperature T and activation energy, Ea (typically 20 kcal/mol). You can predict the stability at room temperature from a plot of ln(k) vs. 1/T and the half-life t(1/2), the time when half of the initial drug concentration degraded. The self-life is the time for 10% degradation (t90) at 25 C (298 K). The Eyring equation provides insight on the mechanism of reaction: entropy, DS‡ (expression of disorder), however, it is less used in preformulation.

Reaction Mechanism

The sequence of the individual elementary reactions is known as reaction mechanism. An elementary reaction involves a few molecules, usually one or two. But why do we need to know the reaction mechanism? By understanding how the API degrades helps design the formulation, it helps in the identification of degradation products, and helps in analytical method development for structural alerts (where you need to go to ppm level quantitation). Often, a compound’s degradation does not stop at the first reaction step as in the case of oxidation. However, of importance for a drug product on stability is the first reaction step. The reaction mechanism can help you determine which this step is; the kinetics gives you an answer about timing.

Hydrolysis, oxidation, thermal and photodegradation, metal ions

Hydrolysis is the most likely cause of drug instability. Water plays a dominant role (in most case is implicated passively, particularly in solid dosage forms), involving nucleophilic attack of liable bonds. The order of reaction is lactam > ester > amide > imine and it is first order. It can be acid and base catalyzed, specific, general (Bronsted), and can involve metal ion mediated reactions. To add more complications, there are buffer catalysis in some cases and a concentration dependence study is needed to rule in/out this process. Since buffers are excipients, more discussions will be allocated to this subject in the following blog.

Oxidation and photo-oxidation is controlled by the environment. It may involve light (UV or visible), trace metal ions, molecular oxygen, and oxygen reactive species. When oxidation is due to molecular oxygen, the reaction is spontaneous at room temperature and is known as auto-oxidation (e.g., rancidity of oils). Oxidation reactions are free radical and chain reactions, the products are colored (pink, amber, brown, black) and sometimes toxic. Oxidation in the solid state is generally insignificant.

The thermal degradation often involves a radical intermediate. Thermal degradation, oxidation and photodegradation occur by a free radical reaction mechanism and sometimes is hard to separate them. If you have a photo-liable molecule, special attention needs to be paid during analytical sample preparation to avoid false conclusions. Manufacturing unit operations also need to be aware about possible degradation upon light exposure.

Take-home Lesson

To stabilize a drug product, you need to know how a drug substance falls apart. Do not cut corners on forced degradation. Important for a drug product on stability is the first reaction step. The reaction mechanism can help you determine which is this step; the kinetics gives you an answer about timing. Hydrolysis is the most likely cause of drug instability. Thermal degradation, oxidation and photodegradation occur by a free radical reaction mechanism.

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