Departments

Call and no. of the operation: Early Career Researchers 2.0; Operation Code (IS e-MA): OP20.01263; Contract number: C3330-17-529013

Name of the beneficiary: National Institute of Chemistry

Participating economic entity: LEK pharmaceutical company d.d.

Project duration: 01.06.2017. – 31.05.2020

Placement in the priority area of the Slovenian Smart Specialization Strategy (S4): Health - Medicine

Program group in which the researcher is employed (title, leader and code): title: Functional foods and food supplements (Institute of Chemistry); head of the program group: dr. Irena Vovk; code: P1-0005 (C)

1. Brief description of the project 

The development and manufacture of a new generic drug requires a large number of steps that can only be performed with quality analytical support based on various analytical techniques. At the same time, the new drug presents a unique analytical challenge, as it is necessary to select an appropriate analytical method to monitor a certain parameter (which in the early stages of development leads to the decision for the next step in development, and in the later stages is crucial for the confirmation of the quality of the drug) which ensures appropriate performance.

The main content of the research project is the development, testing and qualification of analytical methods based on liquid chromatography to determine the quality of active ingredients and drugs that will follow the regulatory trends in the requirements for pharmaceutical products. Requirements for the determination of traces of impurities and potentially genotoxic impurities require the use of modern analytical techniques, with the possibility of determining the minimum amounts of these substances. As part of the project, we will examine the possibility of using certain analytical techniques based on liquid chromatography, which, due to their sensitivity and low limits of detection, will give us a competitive advantage.

Within the research project, the biggest challenge will be the choice of analytical techniques, the development of appropriate analytical methods and also the possibility of performing them in routine product quality control. The ultimate goal is to obtain reproducible and accurate results that reflect the real quality of the newly developed drug at all stages of development and later in its production and quality control.

This will guarantee the high quality and safety of the product, which will ensure the proportion of known impurities below the level for each known impurity and below the level for unknown impurities in accordance with the ICH (The International Conference on Harmonization) guidelines.

2. Project results:

2.1. Impurities migrating from plastic and other labware interfere with pharmaceutical analyses.

During the analyses of a specific active pharmaceutical ingredient (API003), unknown peaks appeared in the chromatogram of the sample that interfered with the analysis, which could consequently lead to misinterpretation of the results. We discovered that the main source of these impurities is plastic labware. The main impurity was oleamide, which is often used as a lubricant and antistatic agent in the polymer industry and is biologically active according to the literature. Using the newly developed LC-MS method, we found that the impurity is leached from almost all plastic laboratory materials into solvents, although these are specified by the manufacturer as compatible. The developed method can be further used for pre-testing of labware in the pharmaceutical industry, in order to select the appropriate one for certain analyses. This will greatly reduce the possibility of misinterpreting the results.

2.2. Human exposure to a bioactive compound that migrates from food/ beverage and medicine containers.

The presence of the bioactive compound (from Section 2.1) has also been demonstrated in plastic food/beverage and medicine containers (e.g. insulin syringes, medicinal syringes, intravenous infusion bottle, enteral syringes) available on the market. Organic solvents and simulants were used as the extraction medium. The human body is therefore exposed to the intake of this compound on a daily basis through ingested food/beverages and medicines, either per os or parenterally, which consequently affects people's exposure and their health. We performed quantitative studies of plastic containers’ extracts and estimated the intake of this compound in the human body. For the purpose of quantitative analysis, the method was validated and the results were statistically evaluated. Recent studies show that this compound can be highly toxic, which was previously unknown, and therefore the introduction of this compound into plastic containers has been allowed without a certain limit by regulatory authorities. These studies will help to improve the regulations for the use of plastic materials and articles intended to come into contact with food/beverages and medicines. The results of this study will certainly lead to intensive toxicological studies in the future.

2.3. A new approach to testing the compatibility of amine APIs with different excipients for early detection of potential incompatibilities between active ingredients and excipients.

To develop a new testing approach, we used LC-MS to analyze a huge number of aged samples arranged by groups (API003 - one set, API005 - one set, API007 - one set, API008 - two sets, API009 - two sets). For this purpose, we developed and validated 5 LC-MS methods. We demonstrated the incompatibility of amine active ingredients with the excipients PEG 6000 (plasticizer) and Fe2O3 (dye) when they are physically separated in formulations (e.g. API in the core and excipients in the over coat) and present in certain proportions. This incompatibility was manifested by an increase in N-methyl and N-formyl impurities. The combination of PEG 6000 and iron oxide was present in 2/3 of the FDA (Food and Drug Administration) approved tablets for 2018. Due to this fact, the usefulness of the developed test will be great. As the test allows the detection of incompatibility of amine active ingredients with excipients at an early stage of drug development, it will reduce the time and cost of performing these analyses in the pharmaceutical industry and will consequently lead to increased competitiveness.

Figure 1: Together with colleagues from Lek d.d., we developed a new approach to testing the compatibility of amine active ingredients with excipients. The test enables the detection of incompatibilities at an early stage of drug development and leads to a reduction in the time and cost of performing these analyses in the pharmaceutical industry, and consequently increases the competitiveness. (Robnik, B.; Naumoska, K.; Časar, Z. A Novel Testing Approach for Oxidative Degradation Dependent Incompatibility of Amine Moiety Containing Drugs with PEGs in Solid-State. Pharmaceutics 2020, 12, 37.)

2.4. Demonstration of unsuitability of the Pharmacopoeial HPLC-UV method for analysis of a particular antibiotic and development of a new NMR method.

We demonstrated the unsuitability of the HPLC-UV Pharmacopoeial method for testing of a particular drug (antibiotic containing the active ingredient API006) by testing the validation parameters. The parameter 'repeatability', for one of the impurities of the active ingredient API006, did not meet the set criteria, for which a new NMR method was then developed. The latter has proven to be superior to the proposed Pharmacopoeial HPLC-UV method and can be further used for quality control of this drug. The new method, through reliable quality control, will enable the reliable control of impurities in the drug and will consequently have an impact on increasing the quality of the drug and on the protection of patients’ health.

2.5. Other work.

Work on the project also included literature review, article writing, theoretical education according to Lek's general procedures, and theoretical-practical training according to Gantt chart. In addition, the work on the project was presented with two posters at the ‘HPLC 2019’ symposium, which was organized in June 2019 in Milan (https://www.hplc2019-milan.org/).
 

3. Bibliographic data derived from the implementation of the project

3.1. Original research papers published in peer reviewed journals

1. ROBNIK, Blaž, NAUMOSKA, Katerina, ČASAR, Zdenko. A novel testing approach for oxidative degradation dependent incompatibility of amine moiety containing drugs with PEGs in solid-state. Pharmaceutics. [Online ed.]. 2020, vol. 12, iss. 1, str. 1-21, ilustr. ISSN 1999-4923. DOI: 10.3390/pharmaceutics12010037. [COBISS.SI-ID 1535326]
2. JUG, Urška*, NAUMOSKA, Katerina*, METLIČAR, Valentina, SCHINK, Anne, MAKUC, Damjan, VOVK, Irena, PLAVEC, Janez, LUCAS, Kurt. Interference of oleamide with analytical and bioassay results. Scientific reports. 7 Feb. 2020, vol. 10, article no. 2163, str. 1-12, ilustr. ISSN 2045-2322. www.nature.com/articles/s41598-020-59093-1, DOI: 10.1038/s41598-020-59093-1. [COBISS.SI-ID 40402949]
(* - shared first authorship)
3. NAUMOSKA, Katerina*, JUG, Urška*, METLIČAR, Valentina, VOVK, Irena. Oleamide, a bioactive compound, unwittingly introduced into the human body through some plastic food/beverages and medicine containers. Foods. May 2020, vol. 9, iss. 5, str. 549-1-549-18, ilustr. ISSN 2304-8158. www.mdpi.com/2304-8158/9/5/549, DOI: 10.3390/foods9050549. [COBISS.SI-ID 13803779]
(* - shared first authorship)
4. MAKUC, Damjan, ŠVAB, Živa, NAUMOSKA, Katerina, PLAVEC, Janez, ČASAR, Zdenko. Determination of D-cycloserine impurities in pharmaceutical dosage forms : comparison of the International Pharmacopoeia HPLC-UV method and the DOSY NMR method. Molecules. 2020, vol. 25, no. 7, str. 1684-1- 1684-15, graf. prikazi. ISSN 1420-3049. www.mdpi.com/1420-3049/25/7/1684, DOI: 10.3390/molecules25071684. [COBISS.SI-ID 1568862]

3.2. Scientific conference contribution abstracts – poster presentations

1. JUG, Urška, NAUMOSKA, Katerina, METLIČAR, Valentina, SCHINK, Anne, MAKUC, Damjan, VOVK, Irena, PLAVEC, Janez, LUCAS, Kurt. Polymeric lubricant, an usually overlooked interference in analytical studies and bioassays. V: 48th International Symposium on High-Performance Liquid Phase Separations and Related Techniques, Milan, Italy, 16-20 June 2019. Milan: [s. n.]. 2019, str. [1]. www.hplc2019-milan.org/pages/programmeAtGlance/searchPopup.html. [COBISS.SI-ID 6677530]
2. NAUMOSKA, Katerina, JUG, Urška, METLIČAR, Valentina, VOVK, Irena. Bioactive polymer lubricant unconsciously administered into the body. V: 48th International Symposium on High-Performance Liquid Phase Separations and Related Techniques, Milan, Italy, 16-20 June 2019. Milan: [s. n.]. 2019, str. [1]. www.hplc2019-milan.org/pages/programmeAtGlance/searchPopup.html. [COBISS.SI-ID 6676762]
 

4. Financing

The investment is co-financed by the Ministry of Education, Science and Sport of the Republic of Slovenia and the European Union under the European Regional Development Fund (ERDF). The public tender for the selection of operations is carried out within the Operational Program for the Implementation of European Cohesion Policy in the period 2014–2020 (www.eu-skladi.si).