Abstract
Background
Stabilis® is an international database on stability and compatibility of drugs. The stability data comes mainly from publications of pharmaceutical journals. As the quality of the published stability studies is not equivalent, the objective of this work was to propose a level of evidence for the physico-chemical stability studies selected for the database.
Methods
At first, we evaluated the main pharmacological class consulted by the users. This work was then divided into 5 steps: (1) updating of the criteria to validate a stability study, (2) creating a grid rating articles, (3) rating of the articles of stability studies for anticancer, antifungal and antiviral drugs by 2 evaluators, (4) creation of new screens in the database to enter rating, to visualize the pictograms and commentaries by the users, (5) creation of a guideline to explain the different levels of evidence.
Results
The main pharmacological class consulted by Stabilis® users is the anticancer drugs and then antiinfectives. We have selected anticancer, antifungal and antiviral drugs for our study. Two hundred and forty publications were evaluated. The highest level attributed was A for anticancer and antifungal drugs and C for antiviral drugs. This difference can be explained by the fact that the majority of publications about antiviral drugs were older.
The most frequent anomalies in the rating of articles were an incomplete or imperfect validation of the analytical method (high value of the coefficient of variation) and a defect in the evaluation of the stability indicating capacity.
It must be noted that the level of evidence is not the quality level of the analytical method but the mixture of the quality of the method validation and of the results. This aspect was a choice of the Stabilis® team and seems important because the security of the patient is impacted by both aspects.
Conclusions
This new function contributes to help the Stabilis® users to evaluate the stability data published and to take a decision for their use in daily practice. This function will be progressively extended to other pharmacological classes of injectable drugs and then for non-injectable preparations.
Introduction
Stabilis® is an international database which presents stability and compatibility studies of medications. It was created in 2001 and is accessible freely on the website www.stabilis.org since February 2008. The originality of the database is that the majority of the information is translated into 29 languages and given by using pictograms.
The stability data given by the manufacturers of drugs are often limited and do not cover the needs for daily practice in hospitals. The majority of the information provided by Stabilis® comes from publications of pharmaceutical journals. These data have been selected by hospital pharmacists of the Stabilis® team by using a standardized document to analyze the publications. The criteria for the selection of an article include especially the type of analytical methods, the validation of the method, the variability of the results, the global coherence of the publication. However, the quality of the selected publications is not equivalent and this aspect was not taken into account for the users of the database.
The objective of our work was to propose a level of evidence for the articles on physico-chemical stability for injectable drugs of three pharmacological classes frequently consulted by the users: anticancer, antifungal and antiviral drugs.
Methods
All stability studies selected for rating were taken into account during a first period for anticancer drugs in 2013 and during a second period for antifungal and antiviral drugs in 2016. To begin this work, we considered only the stability studies devoted to injectable forms for these three pharmacological classes. We decided to select only the physico-chemical stability studies. The other information included in Stabilis® as “incompatibilities” or “factors affecting stability” were not considered in the field of this work because they do not require a complex analytical method. All articles dealing only with visual compatibility of drugs were excluded from this evaluation.
The first step of this work was to update the document created to analyze each scientific publication of the database. This form was carried out by using recommendations of ICH Guidelines Q1A (R2) [1], Q2 (R1) [2], Q1E [3] and the recommendations of the guideline for practical stability of anticancer drugs [4]. Two forms were created, one for classical small molecules and one for proteins (enzymes and monoclonal antibodies). This one was necessary because of the complexity of the biological molecules which need complex analytical methodology to evaluate the stability.
For classical molecules, the following items have been included in the datasheet:
for physical stability
visual inspection (search for a precipitate, change of color, gas formation)
subvisual evaluation (turbidimetry, particle count, microscopic analysis)
for chemical stability, the analytical method should be validated including the stability indicating capacity to separate the degradation products (DP) from the intact molecule. The reference method is the High Performance Liquid Chromatography (HPLC). About the chemical stability, the concentration of the molecule studies at the end of the stability study were noted as more than 90 % of the initial concentration (T90 %) or more than 95 % of the initial concentration (T95 %). Other possible methods were also noted like pH measurement, osmolality, weight evaluation.
For the protein, in accordance with the ICH guideline Q5C [5], we have listed methods used for these complex molecules but this list is non-exhaustive:
for physical stability:
Visual inspection
Turbidimetry to evaluate microaggregates
Dynamic Light Scattering (DLS) to evaluate soluble and non soluble aggregates
Size Exclusion Chromatography (SEC) to show monomer and dimer.
for chemical stability, the methods should appreciate the various structures of the protein (primary, secondary, tertiary structure). Among the used methods:
Cation exchange chromatography
Peptide mapping
SEC can also detect breakage in the protein structure
Capillary electrophoresis
Second derivative UV spectrometry to evaluate changes in the tertiary structure
Second derivative IR spectrometry to evaluate changes in the secondary structure
a biological analysis.
The second step was to create coherent levels of evidence by using the criteria of the datasheet. Two documents have been created, one for classical molecules and one for proteins. These two documents use different criteria but have the same level of evidence. The rating grid for classical molecules is presented in Table 1 and for proteins in Table 2.
Level of evidence for classical molecules.
| Level of evidence A or A+ if the subvisual examination has been performed | ||
|---|---|---|
| A | A+ | – Separative method and complete analytical validation and low variability (CV<5 %) |
| – Chemical stability established with 95 % of the initial concentration (T95 %) and physical examination studied (visual aspect). | ||
| Level of evidence B or B+ if the subvisual examination has been performed | ||
| B | B+ | – Same criteria as level of evidence A but physical stability not performed |
| – OR Chemical stability defined as the concentration above 90 % of the initial concentration (T90 %) by a validated analytical method and small variability. | ||
| Level of evidence C or C+ if the subvisual examination has been performed | ||
| C | C+ | – T95 % but separative method failed because of one or two of the following criteria: |
| – Stability indicating capability insufficiently studied | ||
| – Variability of the results (CV>5 %) | ||
| – Within day or between day reproducibility not precised or out of the specifications (within day repeatability: CV<2 % and between day reproducibility: CV<4 %) | ||
| – Unencrypted results (eg graphic) | ||
| – Separation from degradation products and Internal Standard not or badly evaluated | ||
| – OR method different from HPLC not justified and T95 % | ||
| Level of evidence D | ||
| D | – T90 % but the validation of the separative method with lacking criteria | |
| – OR T95 % but the validation of the separative method includes more than 2 failing criteria. | ||
| – OR method different from HPLC not justified and T90 % | ||
Level of evidence for proteins.
| Level of evidence A or A+ if the subvisual examination has been performed | ||
|---|---|---|
| A | A+ | – Physical examination: at least 2 methods |
| – Chemical analysis: at least 3 methods | ||
| – Biological analysis: at least 1 method | ||
| Level of evidence B or B+ if the subvisual examination has been performed | ||
| B | B+ | Same criteria as level A but without biological analysis |
| Level of evidence C or C+ if the subvisual examination has been performed | ||
| C | C+ | Only 3 methods including physical, chemical and/or biological methods |
| Level of evidence D | ||
| D | Only physical method or less than 3 methods | |
The rating includes 5 levels of evidence from A to D, level A representing the highest level. An additional level E was created for stability studies using only a biological method. Secondary levels have been added (A+, B+ and C+) for stability studies where the subvisual evaluation has been performed. A last form called “Joker” was attributed when more than one molecule have been evaluated in a publication with different levels of evidence depending on the molecules.
In addition to the level of evidence selected, standardized commentaries can be included in the form. They can justify the form or add complementary information which does not influence the form.
Increased concentration unexplained
Stability indicating capacity insufficiently studied
Intra-day or inter-day reproducibility not evaluated or out of the specifications
No visual inspection
No comments for the degradation products
Changes in pH not commented or difficult to interpret
The third step was to use these criteria. Two evaluators were included in this work. In a first phase, 10 articles were analyzed by the evaluators and by two referents. The conclusions were compared. The interpretations of certain criteria were specified to homogenize the results and a new evaluation was carried out before beginning the final form.
The fourth step was to create new screens and new pictograms. The majority of the information in the database is given with pictograms, translated into the 29 languages of the database.
Only articles published in pharmaceutical journals have been selected excluding stability data provided by the manufacturers. Therefore, a special pictogram has been created to show these data. The publications which concern only physical compatibility were not in the field of this work and no level of evidence has been attributed.
The fifth step was to create a guideline to explain what the different levels of evidence are and how they had been built.
Results
Number of publications evaluated
Anticancer drugs in injectable forms are the first pharmacological class consulted by the Stabilis® users. Around 33 % of the users open at least one monograph of anticancer drugs. Their stability studies were therefore the first articles to be evaluated for the form in 2013. Two other pharmacological classes were evaluated in 2016: antifungal and antiviral drugs in injectable forms. They belong to the antiinfective drugs which represent the second center of interest of the Stabilis® users with around 27 % of the users who open a monograph of anti-infective drug. Table 3 shows number of monographs and publications evaluated in Stabilis® for these three pharmacological classes. In Table 3, we can notice that respectively 61.6 %, 19.4 % and 30.7 % of the articles of anticancer, antifungal and antiviral drugs were rated.
Summary of data for anticancer, antifungal and antiviral drugs in the Stabilis® database.
| Number of monographs | Number of publications | |
|---|---|---|
| Anticancer drugs(As of 1st August 2013) | 79 | 318 with 196 publications evaluated (61.6 %) |
| Antifungal drugs(As of 1st March 2017) | 10 | 108 with 21 publications evaluated (19.4 %) |
| Antiviral drugs(As of 1st March 2017) | 5 | 75 with 23 publications evaluated (30.7 %) |
About the antifungal drugs, the majority of stability studies has been carried out for amphothericin B and its various forms (liposome, deoxycholate) (7 studies/21) and also about fluconazole (7 studies/21). About antiviral drugs, stability studies were mainly for aciclovir and ganciclovir.
Comparison between the levels of evidence
The Figure 1 shows the percentage of the different levels for the three pharmacological classes. Level A and A+, B and B+ and C and C+ have been mixed for a better visualization. Level A is the most frequent for anticancer and antifungal drugs. For anticancer drugs, the number of articles decreases with the level of evidence (fewer articles with level B, C and D), which is a witness to the good quality of the articles. For antiviral drugs, the most frequent level is C.
Comparison of the level of evidence in percentage for anticancer, antifungal and antiviral drugs.
For anticancer and antiviral drugs, the majority of stability studies shows a chemical stability with more than 95 % of the initial concentration at the end of the study, as recommended by ICH guidelines and the European guideline for practical stability studies [1, 4], see Figure 2. For antifungal drugs, only 47 % of the stability studies demonstrate the T95 % for the chemical stability.
Comparison of the number of publications defining chemical stability as 90 % of the initial concentration and 95 % of the initial concentration (T95 %) for the three pharmacological classes anticancer, antifungal and antiviral drugs.
On the Stabilis® website, the results of the rating for the articles are visible in the form of pictograms, see Figure 3. In this example, data have the level A+, other data have the level B+ and the last two lines are information given by the manufacturer with a specific pictogram.
The screen “Stability of solutions” of the rituximab monograph.
By clicking on the pictogram of the level of evidence, the user obtains a second screen (Figure 4). This screen shows the level of evidence, the methods used to demonstrate the physical and chemical stability, another method and the commentaries. Forty-two pictograms have been created for this new function, see Table 4.
Example of the screen available for the users after clicking on the pictogram “Level of evidence”. N.B: T95 %: Chemical stability defined as 95 % of initial concentration. HPLC: High performance liquid chromatography. UV: ultraviolet. DAD: photodiode array detector.
Pictograms created for the new database in Stabilis®.
| Pictogram | Meaning | Pictogram | Meaning | |
|---|---|---|---|---|
| Level of evidence |  | Level of evidence A |  | Level of evidence C |
 | Level of evidence A+ |  | Level of evidence C+ | |
 | Level of evidence B |  | Level of evidence D | |
 | Level of evidence B+ |  | Level of evidence E | |
 | Joker |  | Manufacturer’s stability data | |
| Chemical stability |  | Thin Layer Chromotography |  | High Performance Liquid Chromatography – Evaporative Light Scaterring detector |
 | High Performance Thin Layer Chromatography |  | High Performance Liquid Chromatography – Diode Array detector | |
 | Gas Chromatography |  | High Performance Liquid Chromatography – Electrochemical detector | |
 | Nuclear Magnetic Resonance |  | High Performance Liquid Chromatography – fluorescence detector | |
 | Stability defined as 90 % of the initial concentration |  | High Performance Liquid Chromatography – mass spectrometry | |
 | Stability defined as 95 % of the initial concentration |  | High Performance Liquid Chromatography – ultraviolet detector | |
 | Protein: intrared spectrometry and second derivative |  | Protein: ionic chromatography | |
 | Protein: peptide mapping |  | Protein: ultraviolet spectrometry | |
 | Protein: ultraviolet spectrometry and second derivative |  | Capillary electrophoresis | |
 | Ultraviolet spectrometry | |||
| Physical stability |  | Visual examination |  | Particle counting |
 | Microscopic examination |  | Turbidimetry | |
 | Protein: Dynamic Light Scattering |  | Protein: Size Exclusion Chromatography | |
 | Protein: Thermal Aggregation Curve | |||
| Other method |  | Mass balance |  | Search of DEHP |
 | Osmolality measurement |  | pH measurement | |
 | Water loss by evaporation |  | Protein: biological activity | |
 | Protein: ultracentrifugation | |||
Discussion
Rating grid
Our goal was not to attribute a note to the publications. Therefore, the level of evidence was chosen because this rating is recognized in the field of health with the example of the French Health Authority with 3 levels [6]. Our rating grid was created in 2013 for the rating of the anticancer drugs. For this pharmacological class with often a narrow therapeutic index, it seems important to distinguish the stability studies with a chemical stability demonstrated with 95 % of the initial concentration and those with 90 % of the initial concentration at the end of the study. Moreover, as many preparations are carried out for outpatients, the preparation in advance using the Dose Banding concept [7] is more and more used in centralised units of anticancer preparation. In this concept, the dose is rounded at ± 5 % (sometimes more) in comparison with the dose calculated with the body surface area. In this context, only stability studies with a 5 % modification are acceptable.
In many publications, the authors defined stability as “a concentration>90 % of the initial concentration”. However many articles having this definition have results above 95 % of the initial concentration at the end of the study. Our choice was to consider the T95 % in the rating grid instead of the T90 % indicated in the definition given in the article.
The subvisual examination has been included in each level to avoid the creation of too many levels (level A and A+, B and B+ etc).
Anomalies in the analytical methods
The ICH Q2 (R1) [1] describes the validation of analytical methods with notably the intra-day and inter-day repeatability, standard curve. Some articles have high values of repeatability in the validation of the method and sometimes also in the results of the stability study. In the stability studies of antiviral drugs, half of the studies have problems with these criteria.
The stability indicating capacity is a crucial part of the study. The authors have to demonstrate that the analytical method used is stability-indicating. This aspect is validated by stress degradation with the objective to obtain degradation products clearly separated from the studied molecule. This stress degradation should not be too strong (around 20 %) [2]. A too strong degradation produces degradation products of the first DP and the concentration of the molecule studied is often too low. This kind of default is observed in some publications.
Level D
Respectively 16 %, 29 % and 30 % of the articles of anticancer, antifungal and antiviral drugs have been evaluated as level D. Several possibilities can induce a level D. All publications evaluated with level D have not the same profile and the user has to take into account the commentaries associated with the reference.
The most frequently drawback is the stability indicating capacity insufficiently or imperfectly studied with the molecule totally destroyed and no degradation product on the chromatogram or the molecule too much destroyed (80 %) and many degradation products which cannot be seen in real life.
The other problems detected were:
Very high coefficient of variation (CV) during the repeatability or intermediate precision evaluation or very high CV in the results,
Separation of the peak of internal standard from the degradation products not evaluated,
Variation of the pH values without any commentaries from the authors,
Stability defined as 90 % of the initial concentration and around this value during the stability study but no comment about the presence or absence of degradation products on the chromatograms. In this situation, we cannot distinguish a real degradation from an interaction with the container.
Standardized commentaries
The standardized commentaries justify the level of evidence attributed or bring complementary information to the users. These commentaries are presented with text because it was not possible to translate this information with pictograms. The text is translated into 29 languages.
A method called “stability indicating” can detect and quantify the DP. However, in many publications after having demonstrated the stability indicating capacity and identifying the relative retention of the DP, the authors do not give information about the apparition of DP even if the concentration of the molecule studied decreases with time. We have created some commentaries about this topic but they do not influence the level of evidence.
No comments for the degradation products
Degradation products identified
Degradation products not observed in real conditions
Degradation products identified and quantified
Degradation products quantified but not identified
Limits of the evaluation
The restricted choice of pharmaceutical classes: The first choice was for the main class consulted by Stabilis® users, anticancer drugs. The ultimate goal will be to evaluate all articles of stability by 2020.
Some studies have a lower level of evidence because some characteristics were not present in the publication, although performed by the authors, like the precise description of the analytical method. Some authors further send to Stabilis® complementary descriptions of the method that were not published to reevaluate the level of evidence.
Other studies are published by some journals as a letter to the editor instead of the full article with the limitation in space and less detail in comparison with a full article which may under evaluate the rating.
The level of evidence must be clearly understood by the users. The level of evidence is not the quality level of the analytical method but the mixture of the quality of the method validation and of the results. It was a choice of the Stabilis® team. The high quality of the method validation with a stability defined with 90 % of the initial concentration instead of 95 % can give a lower level. This aspect seems important to us because the security of the patient is impacted by both aspects and not only but the analytical method. The concentration of the drug of interest is not the only point to establish stability data. The presence or absence of toxic degradation products should be analysed. A study establishing stability with 95 % or 90 % of the initial concentration but detecting a low concentration toxic degradation product is not retained in the Stabilis® database and the data are not referenced.
Conclusion
The Stabilis® database has been in constant evolution since the opening in 2001. As the quality of the stability studies may be variable, we have created a rating form to propose different levels of evidence for the articles of physico-chemical stability to help the users in their decisions. Four levels have been created (A, B, C and D) and an additional level E for the studies using a biological method.
The first pharmacological class was the anticancer drugs evaluated in 2013 and two additional classes were evaluated in 2014 (antifungal and antiviral drugs). The future objective is to extend this rating to all stability studies of injectable drugs beginning with anti-infective drugs and then for the non-injectable drugs.
About the authors
Elise D’Huart studied pharmacy in Nancy since 2010. She started her hospital pharmacy internship in November 2015 in Nancy. Since 2016, she is a member of STABILIS®, works on research bibliography and performs stability studies.
Pauline Lider is a hospital pharmacist in the University Hospital of Nancy since 2013. Her domains of interest are clinical and oncology pharmacy, reconstitution of cytotoxic drugs and stability studies. She is also a member of INFOSTAB, a French non-profit association, and a member of the STABILIS® team for which she has initiated work on level of evidence.
Jean Vigneron is a hospital pharmacist in the University Hospital of Nancy since 1985. His domains of interest are reconstitution of cytotoxic drugs, analytical control and stability studies.
He is also president of INFOSTAB, a French non-profit association and creator of the international database STABILIS®, dedicated to the stability and compatibility of drugs.
Béatrice Demoré, Ph D, is a hospital pharmacist at the University Hospital of Nancy since 1998. Since 2017 she is head of pharmacy. Her domains of interest are antibiotic drugs and clinical pharmacy. She is also secretary of INFOSTAB, a French non-profit association and a member of the STABILIS® team. Since 2008, she is Associate Professor in clinical pharmacy at the Faculty of Pharmacy of Nancy.
Acknowledgements
Assistance with the study: thank you to Jacques Kuhnlé for reading through it all and making meticulous corrections. Financial support and sponsorship: none.
Conflict of interest statement: Authors state no conflict of interest. All authors have read the journal’s Publication ethics and publication malpractice statement available at the journal’s website and hereby confirm that they comply with all its parts applicable to the present scientific work.
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Article note
Preliminary data was presented as a poster during the European Conference of Oncology Pharmacy in Krakow, Poland, in June 2014 – New function on the Stabilis® database: Level of evidence for stability studies of anticancer drugs. Lider P, Vigneron J, Gindre I, May I, Demoré B. University hospital of Nancy, France.
© 2018 Walter de Gruyter GmbH, Berlin/Boston
