Index (Jump to)

Index of Figures

Index of Tables

1.Introduction

1.1.Forced degradation studies

1.1.1. Overview of regulatory guidance

1.1.2. Review of existing literature and different approaches

1.2.Furagin

1.2.1. Chemical and Physical Information

1.2.2. Pharmaceutical Information

1.2.3. Impurities

Index of Figures

Figure 1: Flow diagram with the different forced degradation conditions to be used for drug substances and drug products (adapted from 16)

Figure 2: Flow chart for performing stress studies for hydrolytic degradation under acid and alkali conditions.

Figure 3: Flow chart for performing stress studies for hydrolytic degradation under neutral conditions.

Figure 4: Flow chart for performing stress studies for degradation under oxidative conditions.

Figure 5: Flow chart for performing stress studies for photolytic degradation.

Figure 6: Furagin

Figure 7: 1-{(E)-[(2E,4E)-5-(5-nitrofuran-2-yl)penta-2,4-dien-1-ylidene]amino}-imidazolidine-2,4-dione

Figure 8: Reaction scheme of impurity A

Figure 9: 1-[(Z)-3-(5-Nitro-furanyl-2)-propene-2-(Z)-ylidenamino]-imidazolidine-2,4-dione

Figure 10: Reaction scheme of impurity B

Figure 11: Reaction scheme of the formation of Furagin from Impurity B

Figure 12: 5-Nitrofuran-2-carbaldehyde

Figure 13: Reaction scheme of impurity C

Figure 14: 3-(5-Nitrofuran-2-yl)prop-2-enal

Figure 15: Reaction scheme of impurity D

Figure 16: Reaction scheme of the formation of impurity D by acid catalysed hydrolysis of Furagin

Figure 17: 1-{(E)-[(5-nitrofuran-2-yl)methylidene]amino}-imidazolidine-2,4-dione

Figure 18: Reaction scheme of impurity E

Figure 19: {(2E)-1-carbamoyl-2-[(2E)-3-(5-nitrofuran-2-yl)prop-2-en-1-ylidene]hydrazinyl}-acetic acid

Figure 20: Reaction scheme of impurity F

Figure 21: Reaction scheme of the formation of impurity F by basic catalysed hydrolysis of Furagin

Figure 22: 1-Aminoimidazolidine-2,4-dione hydrochloride

Index of Tables

Table 1: Potential Adverse Effects of Instability in Pharmaceutical Products

Table 2: Conditions Generally Employed for Forced Degradation

1. Introduction

The investigation in the discovery, exploratory and development phases of drug research shows several bottlenecks which must be minimized in order to bring candidate drugs faster into full-scale production. Among these, impurity profiling plays an important role ¹. In fact, while pharmaceutical industry has been making efforts to reduce the time and cost that it takes to get products to market, the potential for stability and impurity “surprises” that affect the development timeline has increased dramatically ².

From a chemical perspective, the existence of pharmaceutical impurities are inevitable because no chemical reaction has 100% selectivity – and no chemical compound is “rock” stable ³. Some potential adverse effects that happen as a consequence of this instability can be predicted and are given in Table 1 ⁴.

Table 1: Potential Adverse Effects of Instability in Pharmaceutical Products

As easily understood, changes in drug stability can risk patient safety by formation of a toxic degradation products or deliver a lower dose than expected. Therefore, it is essential to know the purity profile and behaviour of a drug substance under various environmental conditions ⁵.

This crucial information is gathered by testing the stability of the drug product, being part of each phase of the drug development as it is required the time period that the drug product continues to maintain its specification ⁶. The development of a stability indicating method (SIM) is essential to accurately measure the changes in active ingredients concentration without interference of other degradation products, impurities and excipients ⁷. In this field, forced degradation studies play an important role as they demonstrate the specificity of the developed method to measure the mentioned changes when little information is available about potential degradation product ⁸.

Forced degradation studies, stress testing, stress studies, stress decomposition studies, accelerated stability or forced decomposition studies are different ways to refer the same stability tests mentioned ^2,9. Although these studies have been in practice in industry for a long time, they just became a formal regulatory requirement with the introduction of the International Conference on Harmonization (ICH) guideline entitled “Stability Testing of New Drug Substances and Products” (Q1A) in 1993 ¹⁰.

1.1. Forced degradation studies

It is not easy to define what a forced degradation study is. It has become somehow an “artful science”, as it is highly dependent on the experience of the company and of the individuals directing the studies ². In addition to this, many terms have been used interchangeably, like “forced degradation” and “accelerated stability” ².

In 1980, Pope defined accelerated stability testing as “the validated method or methods by which product stability may be predicted by storage of the product under conditions, which accelerate change in a defined manner.” ². With the term “validated”, Pope meant that the change occurring under the accelerated conditions must be demonstrated to correlate with normal long-term storage. More recently, the International Conference for Harmonisation (ICH) introduced an important distinction between “forced degradation” and “accelerated stability” in the context of pharmaceutical stability, pointing “accelerated testing” as ¹¹:

• Studies designed to increase the rate of chemical degradation or physical change of an active drug substance or drug product using exaggerated storage conditions as part of the formal, definitive, storage program. These data, in addition to long-term stability studies, may also be used to assess longer-term chemical effects at nonaccelerated conditions and to evaluate the impact of short-term excursions outside the label storage conditions such as might occur during shipping. Results from accelerated testing studies are not always predictive of physical changes.

And “stress testing” as:

• Studies undertaken to elucidate the intrinsic stability of the drug substance. Such testing is part of the development strategy and is normally carried out under more severe conditions than those used for accelerated testing.

It is clear that there is a differentiation between both concepts, being stress testing distinguished by both severity of the conditions and the focus or intent of the results.

During a forced degradation test, the following conditions should be investigated not only in the drug substance but also in the drug product: acid and base hydrolysis; hydrolysis at various pH; thermal degradation; photolysis; and oxidation ¹².

Forced degradation studies are designed to generate product-related variants and develop analytical methods to determine the degradation products formed during accelerated pharmaceutical studies and long-term stability studies. Any significant degradation product should be evaluated for potential hazard and the need for characterization and quantification ^13,14.

There are many outcome that must be collected throw the forced degradation studies ¹⁵. Among them, we may emphasize:

• Estimation of the stability of the drug substance and drug product;
• Identification of structural transformations of the drug substance and drug product;
• Detection of any low concentration of potential degradation products;
• Detection of unrelated impurities in the presence of the desired product and product-related degradants;
• Separation of product-related degradants from those derived from excipients and intact placebo;
• Elucidation about the possible degradation pathways;
• Identification of degradation products that may be spontaneously generated during the storage;
• Facilitation of improvements in the manufacturing process and formulations in parallel with accelerated pharmaceutical studies

Although we cannot albeit the importance of the stability testing in the safety of the population, there is no standard procedure about how we should carry a forced degradation test is available.

Questions that seem simple, like “How much stressing is enough?” are subject of much discussion amongst pharmaceutical scientists. Despite a value from 5% to 20% of degradation have been considered as reasonable and acceptable for validation of chromatographic assays, some products are so stable that little or no degradant is detected. Furthermore, if we consider expose the product to energy in excess of the energy provided by accelerated storage, it may lead to overstressing and aberrant results ^16–18.

1.1.1. Overview of regulatory guidance

ICH have described forced degradation studies in various international guidelines agreed between American, European and Japanese regulatory authorities and followed by other regulatory authorities ¹⁵. Among them, we may point:

• ICH Q1A(R2) – Stability Testing of New Drug Substances and Products ¹¹;
• ICH Q1B – Stability Testing: Photostability Testing of New Drug Substances and Products ¹⁹;
• ICH Q2(R1) – Validation of Analytical Procedures: Text and Methodology ²⁰;

The ICH Q1A(R2), in section 2.1.2, reports the conditions for performing forced degradation studies on drug substances and products. This test is likely to be carried out on a single batch of the drug substance and include the effect of temperature (in 10°C increments (e.g., 50°C, 60°C, etc.) above that for accelerated testing), humidity (e.g., 75% relative humidity or greater) where appropriate, oxidation, and photolysis on the drug substance. The susceptibility of the drug substance to hydrolysis across a wide range of pH values should also be evaluated when in solution or suspension. The examination of the degradation products under stress conditions is useful in establishing degradation pathways and developing and validating suitable analytical procedures. Although, if it is demonstrated that certain degradation products are not formed under accelerated or long term storage conditions, there is no need to examine them.

The ICH Q1B contains recommendations for assessing the photo stability of drug substances and drug products, being the conditions mentioned in Section 2 and Section 3, respectively. No specific exposure level are defined but it is stated that “samples should be exposed to light providing an overall illumination of not less than 1.2 million lux hours and an integrated near ultraviolet energy of not less than 200 watt hours/square meter to allow direct comparisons to be made between the drug substance and drug product.”. Despite the design of the study is left for the applicant, scientific justification must be given if the light exposure studies are terminated after a short time – may be related with the observation of excessive degradation product. Solid and solution/suspension may be subjected to the photo stability tests, being both used in the development of a stability indicating method.

The ICH Q2 (R1) gives guidance on how to validate analytical methodology and in section 1.2.2 of Part II there is a recommendation to used samples from forced degradation studies to prove specificity when there are no impurities available.

Forced degradation studies where not a regulatory requirement, although carried by the pharmaceutical industry a long time ago, until the introduction of the ICH guideline entitled “Stability Testing of New Drug Substances and Products” (Q1A) in 1993, the precursor of the referred version. Despite the sooner the stress tests are held the easier is the selection of stability-indicating methods, guidelines does not explicitly require its performance or report at the phase 1-2 investigational new drug (IND) stages ⁷.The guidance require drug substance stress testing for Phase 3 IND but the regulatory authorities, however, ask questions concerning the stressing results as early as a Phase 1 IND and suggest this studies in drug products ².

For a New Drug Application (NDA), the guidance require a summary of drug substance and drug product stress studies including elucidation of degradation pathways, demonstration of the stability-indicating nature of analytical methods, and identification of significant degradation products ²¹.It is also required to stress the drug under hydrolytic, oxidative, photolytic and thermolytic conditions in solutions and in the solid state, as previously mentioned.

In the rest of the world, as there are many different regulatory guidance, there are also some considerations that should be stated ⁹:

• Canada follow ICH guidelines with minor differences ²²;
• In South America, guidelines issued by Brazil and Mexico do not explicitly use the word stress testing or forced decomposition, focusing primarily in long-term and accelerated storage. Although, degradation products are emphasised ^23,2424;
• In Central America and Caribbean, Panama, El Salvador, Guatemala, Nicaragua, Costa Rica and Honduras have guidance similar to Mexico and Brazil ^25,26;
• In Africa, South Africa has adopted the ICH guidelines with minor modifications ²⁷. Republic of Kenya has a mix of ICH, EMA and WHO guidelines ²⁸. In Tanzania, one of their guideline clearly states that when we want to do some changes in registered products, documentation should include “The stability profile including the results on stress testing” ²⁹;
• In Golf Cooperation Council, which comprises Bahrain, Kuwait, Oman, Qatar, Saudi Arabia and the United Arab Emirates, the guidelines are parallel to WHO 2009 guideline ³⁰;
• Saudi Arabia define the requirements very similarly to ICH QA1(R2) ³¹;
• The Association of Southeast Asian Nations, which includes Indonesia, Malaysia, Philippines, Singapore, Thailand, Brunei, Cambodia, Laos, Myanmar and Vietnam, describe the stress condition as “40° C±2°C/75% RH ± 5% or at more stressful conditions” ;
• In Singapore, stress testing is regulated as a necessity for various steps of the drug development ³²;
• In India, the requirements are very similar to the ones stated by ICH ³³;
• Australia and New Zealand guidelines are also similar to ICH Q1A(R2) ⁹.

1.1.2. Review of existing literature and different approaches

As the drug degradation pathways differ from drug to drug, it is difficult to decide on the stressed conditions to be employed for a new drug at the time of the initiation of forced degradation studies ³⁴. This can be understood if we think about the Arrhenius relationship – probably the most commonly used expression for evaluating the relationship between rates of reaction and temperature for a given order reaction – as a way to analyse the drug decomposition. If a drug obeys to the Arrhenius relationship, it is possible to estimate the effect on temperature on the degradation rate of the compound, assuming that we know the energy of activation (E_a) ³⁵.

In 2011, MacFaul et al. studied the kinetics of degradation of 166 drug-like compounds in solution at elevated temperatures showing that the mean E_a was 98.6 kJ/mol, within a range from 49.8 kJ/mol to 197.5 kJ/mol ³⁶.

Data from solid-state degradation studies of more than 50 compounds in 100 studies at Pfizer using the Accelerated Stability Assessment Program (ASAP) approach indicated an average E_a of 124.7 kJ/mol ³⁷.

This information show how different drugs are and how difficult it is to come up with a standard procedure for the stressed degradation studies and different approaches have been used by pharmaceutical industry the last years ³⁸.

The design of these studies must use more strenuous conditions than those used for accelerated studies (25°C/60% RH or 40°C/75% RH). At a minimum, acid and base hydrolysis, hydrolysis at various pH, thermal degradation, photolysis, and oxidation must be investigated ²¹. Figure 1 can be used as a guide.

Figure 1: Flow diagram with the different forced degradation conditions to be used for drug substances and drug products (adapted from ¹⁶)

Some authors have tried to emphasize how different are the conditions that drugs are subjected to during forced degradation studies and the uncertain answer we have for some crucial questions. S. Singh published in 2000 some guidance ³⁴ trying to answer the following questions:

• To what extreme of conditions one should go if a previously tested condition does not give sufficient degradation, good enough for degradation products to be isolated in quantity suitable for characterisation and structure elucidation?
• Are there any limits where one should stop and carry no further studies?
• What sort of reagents or agents need to be employed for creating a particular stress condition?

As stated, each drug has a different approach to its forced degradation studies and its degradation shows different rates. Although, the generally recommended degradation varies between 5-20%. This range covers the generally permissible 10% degradation for small molecule pharmaceutical drug products, for which the stability limit is 90%-110% of the label claim ⁵.

Although some scientists have found it practical to begin at extreme conditions (80°C or even higher, 0.5N NaOH, 0.5N HCl, 3% H₂O₂) ³⁹, the conditions generally employed for forced degradation studies are the ones presented in Table 2, published by George Ngwa ¹⁶,because, as previously stated, overstressing can lead to aberrant results ^16–18.

Table 2: Conditions Generally Employed for Forced Degradation

There are some other information that we should take into account concerning the following topics ³⁴:

• Drug concentration of the reaction solution

Some authors, according to their experience, recommend to use the concentration of 1 mg/mL. Although, in certain cases, the studies must be carried at the concentration of the final formulation because the polymerization plays and important role in the final concentration of the drug product. As an example we have aminopenicillins and aminocephalosporins commercial preparations, where a range of polymeric products have been found to be formed when the drug is in high concentrations ⁴⁰.

• Handling of the reaction samples for the chromatographic studies

Sometimes it is difficult to know the best way to handle samples that have a high concentration of acid, alkali or an oxidizing agent for injecting them into HPLC or loading on a TLC plate.

There are two main approaches to deal with this situation: dilution and neutralization.

In the first one, it is advised that we should dilute the sample enough so that the concentration of reagent falls within the tolerable range. In HPLC, the dilution can be done in the mobile phase while using TLC other solvents can be suitable (methanol, ethanol, etc.).

The second one, involves the neutralization of the acid and alkali solutions to a tolerable pH. Although, this has some drawbacks such as the difficulty to carry the experiment out in a quantitative manner and the precipitation of the dissolved ingredients that sometimes occur.

As obvious, the recommended approach is the dilution, as it eliminates the drawbacks of the neutralization. If there is any problem with the dilution, it can be generally solved by increasing the volume of injection or loading. This increase should be guided by the buffer capacity of the buffer used in the mobile phase.

• The design of the studies

For every stress study, it is advised to generate four samples and report the results of each: the blank solution stored under normal conditions, the blank subjected to stress in the same manner as the drug solution, the zero time sample containing the drug which is stored under normal conditions and the drug solution subjected to stress treatment. A real assessment of changes is only made through the comparison of all these results.

• The equipment in use

There are two important equipment for stress testing: the container in which the reaction is done and the equipment for creating the stress conditions. Each may vary according to the reaction that is under studying (hydrolytic, oxidative or photolytic).

For hydrolytic studies in dilute acid and alkali conditions at temperatures between 5° C above room temperature up to 70° C, the reactions can simply be carried out in containers like volumetric flasks or stoppered culture tubes and stored in a water bath set at the desired temperature.

For reactions above 80° C and reflux conditions, one option is to use a boiling water bath equipped with a voltage regulator. Alternatively, an oil bath with a voltage regulator may be employed.

The oxidative stress studies are suggested under normal laboratory conditions only. Hence no specific equipment is needed for the purpose. The studies should be done in a leak proof stoppered containers.

For photolytic reactions, the advice is to follow ICH guidelines. Hence any of the different kind of lamp sources defined in the guideline might be used. The output of the lamp should meet D65/ID65 emission standards defined by ISO. The guideline suggests use of artificial daylight fluorescent lamp combining visible and UV outputs, xenon or metal halide lamp. Singh also provided some useful information for scientists who are not sure how to conduct their forced degradation studies: decision trees with the conditions to which the drug must be subjected in case of hydrolysis, oxidation and photolysis (Figure 4 and Figure 5) ³⁴. The conditions for the study of the effect of temperature and humidity and photostability are already detailed in the ICH guidelines ^11,19.

Figure 2: Flow chart for performing stress studies for hydrolytic degradation under acid and alkali conditions.

Figure 3: Flow chart for performing stress studies for hydrolytic degradation under neutral conditions.

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