Monday, 04 December 2017 08:09

Working group – a Portrait: Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Department “Drug Delivery” Featured

InVitro+Jobs presents scientists and their innovative research in a regular feature called “Working Group – a Portrait”. We focus on newly developed methods, their evaluation and their potential for reducing and where possible replacing animal experimentation according to the 3R principles of Russel & Burch (reduce, refine, replace).
In this issue we present the Department “Drug Delivery” at the Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) headed by Prof. Dr. Claus-Michael Lehr, Professor for Biopharmaceutics and Pharmaceutical Technology at Saarland University.

Working group – a Portrait:
Helmholtz Institute for Pharmaceutical Research Saarland (HIPS),
Department “Drug Delivery”

In light of the increasing threat posed by infectious diseases, the team investigates the properties of biological barriers in the human body and how they can be overcome, so as to deliver drugs to the site where they should ultimately take effect. Biological barriers protect the body against harmful environmental influences, but also selectively allow substances to pass that the body needs (1). The barriers include the skin, the intestine, the oral mucosa and the lungs.

The team develops in vitro methods for use in their research and the scientists believe that only human-specific test methods can lead to reliable and safe pharmaceutical developments.

Image 1: Department “Drug Delivery” headed by Prof. Claus-Michael Lehr at the Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS).
Source: HIPS

The goal is to develop new pharmaceutical drugs, especially antiinfectives and vaccines. This is important, as there is a lack of drugs effective against such diseases. The number of pathogens resistant to common drugs is also on the rise (2).  

Prof. Lehr researches targeted drug delivery to the site of the disease. On the one hand the properties of the biological barriers themselves (skin, lungs and intestine) are studied. For example: The bronchial mucous membrane allows gases and minute particles to pass easily, but stops larger particles such as pharmaceuticals. In order to assess the time needed for an inhaled drug to overcome this barrier, mathematical models are also used. In cooperation with the working group headed by Prof Christian Wagner (Physics, Saarland University), a new mathematical model was developed, which was able to predict that it would take a spherical nanoparticle smaller than 30 to 40 nm in diameter 15 minutes to penetrate the mucus (3).

Image 2: Nano-ferry shaped like a corn cob.
Photo: Marc Schneider/cc-NanoBioNet/German Association of Nanotechnology

On the other hand research is conducted into transport systems suitable for overcoming the aforementioned barriers and delivering the drug to its target site. For example: As nanoparticles get stuck in the rigid, thick gel rods in the lung mucus, as if they were the bars of a cage, the scientists have come up with nano-ferries such as the one shown here that looks like a corn cob (image 2). The tube is 10 µm long and the globules attached to the outside have a diameter of 800 nanometres. These are to later have drugs applied to them, which can then get past the lung mucus (4).

Prof. Claus-Michael Lehr’s research team develops models which can be used for studying the mechanisms of infectious diseases and testing drugs, such as a new human alveolar cell line that with its blood-brain-barrier properties can recreate the in vivo situation in humans. The alveoli are the lung sacs responsible for the gas exchange in the lung (see box below), but can also pass minute particles into the bloodstream. Alveoli are made up of two cell types, type I and type II. The model developed here is made up of type I cells, responsible for creating the barrier between the blood and the air in the lung (see illustration 5). The team was able to immortalise this cell type with the aid of a lentivirus, thus creating a cell line that can be used for testing the effects of inhaled substances  that have already overcome the more superficial bronchial epithelial barrier with mucus and cilia (5). In the tests the cells’ so-called transepithelial resistance is measured.  Insights into the mechanisms in deeper lung layers are urgently needed if substances or drugs are to be able to cross this barrier (6).


Image 3: After transport, drug release and stability experiments with in vitro lung models chromatographic and mass spectrometric investigations are also be conducted.
Foto: HIPS/Bellhäuser.

Inhalable drugs, which cross the called blood-air-barrier into the bloodstream, are definitely attractive for developers of pharmaceutical drugs: In comparison to other organs, the lung has a relatively low enzyme activity. They can also bypass the enterohepatic circulation, in which a substance is metabolised, either detoxifying it or even making it toxic in the first place, leading to undesired side effects (1).

The research is still in its early stages. The scientists still know too little about where inhaled drugs and their carrier particles end up, whether they actually pass through the alveolar epithelium or even reach specific areas or cells in the lung at all.

Prof. Lehr is the recipient of numerous awards, including the Animal Welfare Research Award of the German Federal Ministry of Food and Agriculture (2011) and the Animal Welfare Research Award of Rhineland-Palatinate (2011), which was conferred on him and his team for the development of a chronic inflammatory intestine model in a Petri dish.  In 2015, 2016 and 2017 the journal Medicine Maker named him one of the hundred most influential experts in the field of drug development three years running.

In 1998 he founded with Dr. Eleonore Haltner the spin-off enterprise Across Barriers, which now employs 36 staff. They provide studies for the pharmaceutical, cosmetics and chemicals industry using in vitro models of human-specific cell and tissue systems that can simulate the transport of substances and formulae across biological barriers. He is also co-founder and CEO of the company PharmBioTech GmbH founded in 2010 in Saarbrücken, a not-for-profit subsidiary of Saarland University. He has been head of the department “Drug Delivery” at the Helmholtz Institute for Pharmaceutical Research Saarland since 2009.

The lung: supply and removal organ

The body obtains energy by oxidative degradation of nutrients and therefore needs a constant supply of oxygen (7). This is the lungs’ task. At the same time they take care of removing metabolic end products, especially CO2. Another important task is the removal of foreign particles from the lung (barrier function).


Image 4: The human respiratory tract.
Illustration: LadyofHats, Wikipedia.

The respiratory tract comprises the nose, throat, trachea, left and right primary bronchi, ending in the left and right lungs (8). The trachea and bronchi are lined by a mucous membrane and cilia.
The respiratory epithelium is made up of pseudostratified epithelial cells (a single layer of cells with nuclei aligned in different planes) and lines most of the respiratory tract. On the interior surface of the cells (toward the bronchial lumen) are hair-like cilia. In the epithelium are also so-called goblet cells, secretory cells between the normal epithelial cells that produce mucin, which is secreted to the epithelial surface. This mucous coating provides a protective layer that acts as a barrier against bacteria and particles (9). The mucous layer helps to prevent the passage of particles. Cilia normally sweep particles back out of the respiratory tract. There are also endocrine cells that secrete serotonin and peptides, with which they influence the rate of mucous secretion and cilial motility (10). So-called club cells secrete proteins that help to regulate inflammatory reactions. The lung also has immune cells: alveolar macrophages ingest foreign substances that have advanced to the alveolar region (1, 11). these filled alveolar macrophages are then expelled from the lung with the mucus (10). The lung’s surface is also colonised by microorganisms, the so-called microbiota, which can be altered by environmental influences and affects not only the immune defence (12, 13) but also the metabolism of foreign substances. The alveoli are made up of a single layer of type I and type II alveolar epithelial cells. Inhaled air is initially simply channelled through a continually bifurcating tubular system, at the end of which are the alveolar ducts, which are the actual site of respiration, with the gas exchange in the alveoli taking place by means of diffusion (7).

Type I cells form a thin layer of cells in the alveoli, bordering on the cells in the walls of the pulmonary capillaries. They make up the blood-air barrier, where oxygen and carbon dioxide diffuse. Type II alveolar cells produce pulmonary surfactant, comprising lipids and proteins that reduce surface tension and prevent the alveoli collapsing and adhering during exhalation (14).

Image 5 left: Alveoli with capillary network. Illustration: Alexandr_Mitiuc,
Image 6 right: Longitudinal section through an alveolus. Gas exchange in an alveolus. Illustration: designua


Prof. Lehr develops in vitro models with human cells for both the bronchial epithelium and the alveolar epithelium with the goal of being able to treat lung diseases with new drugs, something that is urgently needed.

Lung diseases on the rise
Lung diseases belong to the main causes of deaths following heart attacks and strokes worldwide. Especially pneumonia, chronic-obstructive lung disease (COPD), lung cancer and tuberculosis play a part. These diseases are considered to be responsible for one tenth of all cases of death in Europe in 2008 (15).

There are genetically determined diseases such as mucoviscidosis or alpha-1 antrypsin deficiency (a). However, many lung diseases such as asthma, chronic infections or COPD result from many years of occupational exposure and an unfavourable lifestyle (smoking). A combination of genetic predisposition and environmental influences can lead to COPD or pulmonary fibrosis. Researchers say that a third of all cases of COPD can be attributed to air pollution, which not only affects developing or newly-industrialised countries.

In Europe there is especially increased occupational exposure to the salt di-isocyanate (16), which is used for instance for producing, und beryllium, which is used as an alloy with copper or other metals for manufacturing materials (17). In certain cases fine beryllium dust can lead to berylIiosis or “chronic beryllium disease” (CBD) with impaired lung function (18).

The developing, most dangerous mechanisms are oxidative stress as well as local and systemic (b) inflammatory processes in the lung. After inhalation and adsorption of toxic sulphate, nitrate or metal particles, reactive oxygen molecules are formed in the lung cells. Particles and highly oxidative gases such as ozone and nitrous oxide can trigger local reactions in the lung that lead to oxidative stress. Local and systemic inflammation reactions are triggered by cytokines and chemokines in the body’s own immune system. The result of progressive tissue remodelling with loss of architecture is a pulmonary fibrosis. A sustained inflammation stimulus causes irreparable damage and loss of function. The cause is thought to be an uncontrolled production of the cytokines IL13 (19), which occurs in allergic reactions. IL13 is also suspected to be the main mediator for asthma attacks (20).

Countless animal experiments are conducted in both toxicity testing and the development of pharmaceutical drugs
Studies of the toxicity of gases and dusts primarily use rats. The laboratory animals used in basic or translational research are mostly mice. Basic and applied research seek potential treatments for acute lung damage, pneumonia, pulmonary fibrosis and lung tumours (21).

Toxicity testing

Toxicity testing of gases and dusts is legally required in accordance with the OECD Test Guidelines 403 (Acute Inhalation Toxicity), 433 (Acute Inhalation Toxicity: Fixed Concentration Procedure), 436 (Acute Inhalation Toxicity – Acute Toxic Class Method), 412 (Subacute Inhalation Toxicity, 28-day Study) and 413 (Subchronic Inhalation Toxicity, 90-day Study). The study results serve to assess the risk involved with handling the substances (occupational safety and health), a classification of the substances (chemicals) and their labelling on packaging and safety data sheets.

There are numerous developments of human cell cultures or organ-on-a-chip models for toxicological studies around the world, and in individual cases even the entire human respiratory tract has been reproduced in vitro (22). Immune cells have also already been integrated (23, 24) and some developers have already conducted validation studies (25) or are in the middle of such studies (26).


However, no procedure that replaces experiments on animals has been approved and implemented into law so far. It is argued that cell systems cannot for instance reproduce the complex interaction between the lungs and other organs or of biochemical, physiological, anatomical processes und previously determined biomarkers in blood or sputum. In short: Inhalation affects the body systemically.

Replacement methods for inhalation tests on animals thus need not only a reproduction of the lung but also one or more simulated mini-organs and a functioning blood circulation, into which the substance is introduced via the alveoli.  Such a system, known as “human-on-a-chip”, is currently the subject of intense development, for instance by TissUse in Berlin.  

As testing for inhalation toxicity must include both local and systemic tests, participants of a workshop held in the USA recommended building a database with the acute systemic toxicity data already available, compiling a review of the newest toxicity mechanisms and test methods, optimising the in vitro assays and drafting standard protocols for their worldwide application in laboratories (27). 

As current developments are not yet sufficient, regulatory authorities have at least attempted to reduce the use of animals. In the USA and Canada the suggestion was made to completely forgo animal experiments in certain cases in the area of acute toxicology, the so-called Six Pack (acute oral toxicity, acute dermal toxicity, acute inhalation toxicity, primary eye irritation, primary dermal irritation, and dermal sensitisation), and replacing them where possible with animal-free test methods. There have now been several meetings at the OECD level and a Guidance Document was drafted, stating in which cases the tests can be forgone (28).

The Journal Applied In Vitro Toxicology will soon publish a special issue featuring the implementation of alternative approaches for inhalation toxicity testing (29).

Disease and drug research (basic and applied research)

Animal testing is still used for research into lung diseases and possible treatment methods. One example is the so-called A/J mouse. The inbred mouse strain is widespread and used in many areas of research, such as instance in carcinogenity tests, as it is prone to developing lung tumours (30), or zur for researching COPD or pulmonary emphysema, as A/J mice develop an emphysema much more quickly than other strains of mice after exposure to cigarette smoke (31).  However, the increasing trend towards so-called disease-on-a-chip models is inevitable.

Researchers develop cancer models for in vitro disease research, for instance a three-dimensional lung cancer model (32) combining human bronchial cells obtained from patients with connective tissue cells and adenocarcinoma cells from the lung. The small tumour deposits that are formed work their way through the healthy tissue over the course of time, allowing possible therapy options to be studied. In another model, in which healthy human bronchial epithelial cells are genetically altered, individual early stages of cancer development can be investigated (33). A multi-organ-on-a-chip with lung, liver and heart has also already been developed, for studying the organoid systems’ interactions resulting changes to the effects of pharmaceutical drugs (34).


Some pharmaceutical manufacturers have supposedly even equipped whole departments with organ-on-a-chip systems, albeit not to replace animal experiments but rather reduce them by eliminating unsuitable substances better in advance and not taking them into pre-clinical testing on animals.

Researchers like Prof. Lehr and their model developments have been able to play a significant part with regard to persuading the industry to rethink animal testing.

We spoke with Prof. Claus-Michael Lehr about the in vitro models in pulmonary research and his assessment of the prospects for animal-free methods using organ-on-a-chip-systems

InVitro+Jobs: Professor Lehr, you once said that animal testing is not necessarily most suited for your area of research. Why not?

Prof. Lehr: First it must be said that animal experiments are legally required in certain phases of drug development and can’t simply be left out as long as there are no validated methods available with which they can be replaced. On the other hand, the transferability of results from animal experiments to humans is often very poor. This makes it very important to research such alternative methods – not only for legal reasons or animal welfare motives, but also in light of the faster availability of better and safer drugs for seriously ill patients.

InVitro+Jobs: Applicants for permission to conduct animal experiments often argue that the questions they research can only be studied in the organism as a whole. why is that not necessary in your case?

Prof. Lehr: Problems of drug delivery typically arise at certain biological barriers. If they can be reproduced in a suitable fashion in a “test tube”, you can certainly have an advantage compared with the highly complex “black box” an entire organism represents, especially because you can also cultivate human tissue for many barrier models, which means the transferability of such results from animals to humans is no longer an issue.

InVitro+Jobs: Do we already know enough about the human lung to be able to simulate it?

Prof. Lehr: The lung’s physiology is highly complex and not yet as well understood as for example that of the skin or the intestine. Nonetheless there has also been considerable progress developing has in vitro models in this area in the past years.

InVitro+Jobs: You develop alternatives to animal experiments for inhalation testing. How many have you developed and what can you study with them?

Prof. Lehr: It will take a very long time before a possible test system resulting from our research work is available as an actual alternative method and can actually make certain animal experiments superfluous. Nevertheless, one can already gain a great deal of information about a new drug using current models. This shortens development times, avoids unnecessary failures and thereby certainly also reduces a number of animal experiments.

InVitro+Jobs: When are certain models needed?

Prof. Lehr: This depends entirely on the issue at hand. Take the lung for instance: Is it about optimising the placement of inhaled drugs in the lung, their subsequent absorption via the blood-air barrier or the clearance from the air in the lung by bronchial mucus and cilia or macrophages? Each of these questions basically requires a different, unique model.

InVitro+Jobs: You mainly work in the field of pharmaceutical drug development. Can the models also be used for toxicity testing (inhalation toxicology)?

Prof. Lehr: As the difference between a medicinal drug and a poison is only a question of the dosage, that is of course fundamentally possible. However, one must differentiate when testing drugs whether it’s about proof of efficacy or toxicological safety. In the case of cosmetics and chemicals it’s essentially only about the latter aspect.

InVitro+Jobs: There are many models culminating in complicated organoid systems on a chip or ones capable of respiration-like movements. Can you assess why such models have not yet been approved by law?

Prof. Lehr: There are different reasons for that. For one thing – with all due respect for and in part even admiration of the scientific achievement behind them – are still affected by many shortcomings. Secondly, once these shortcomings are seen to be satisfactorily remedied, you have to prove scientifically that the data obtained using such models is reliable. This “validation” is very time-consuming, laborious and expensive. Thirdly, it is definitely debatable how high the hurdle must be for regarding a certain as actually equivalent to an animal experiment, whilst the animal experiment itself basically only represents an estimate of the actual effect on humans, which is what really interests us. Ultimately it is therefore not only a purely scientific question but also about political and social questions; and when it comes down to it, not only ethical aspects but also economic considerations play a role– as is the case for instance with the greenhouse effect and CO2 emissions.

InVitro+Jobs: A company in Geneva has now effectively developed a test tube version of the entire respiratory tract, from the mouth to the alveoli. Is that the way ahead?

Prof. Lehr: Evidently an exciting approach with sophisticated technology that is fascinating in its own right. It remains to be seen if it will be practicable at the end of the day.

InVitro+Jobs: How complicated do the models have to be? When could simple models be sufficient?

Prof. Lehr: They certainly should always be as simple as possible and only as complicated as necessary. However, it is especially difficult assessing the latter when you basically haven’t completely understood the system you are trying to build a model of. We always have to remind ourselves that we are still far from building a living organism, an organ, a tissue or even a single living cell.

InVitro+Jobs: A research team recently developed a proposal for an adverse outcome pathway for respiratory sensitisation. They studied low-molecular chemicals, which also include pharmaceutical drugs. They recommended the development of a test strategy, to some extent using skin sensitisation methods as a frame of reference. What do you think of this approach. Could it also be a viable approach for your future work?

Prof. Lehr: That is certainly a rational approach, which I can relate to very well.

InVitro+Jobs: There are scientists who are required to provide animal experiments as an additional prerequisite for having their research published. Have you ever experienced that yourself?

Prof. Lehr: Yes, some renowned journals have a certain bias in that they only regard the results of research on new drugs as sufficiently relevant when they are obtained by means of animal testing. That is on the one hand intuitively understandable, but on the other hand the results from animal experiments may not necessarily be as transferable to patients’  clinical situation as the results of certain in vitro models based on human cells and tissues.

InVitro+Jobs: Is it more difficult to get funding if you want to work with cell culture models and computer simulations?

Prof. Lehr: Fortunately there is definitely public funding, foundations and even awards for research into alternative or replacement methods for animal testing – quite rightly so for the aforementioned reasons.

InVitro+Jobs: Thank you for the interview.



(1) Gordon, S., Daneshian, M., Bouwstra, J. et al (2015): Non-Animal Models of Epithelial Barriers (Skin, Intestine and Lung) in Research, Industrial Applications and Regulatory Toxicology. ALTEX 32 (4): 327-378. DOI:


(3) Matthias Ernst, Thomas John, Marco Guenther, Christian Wagner, Ulrich F. Schaefer, Claus-Michael Lehr (2017): A Model for the Transient Subdiffusive Behavior of Particles in Mucus. Biophysical Journal 112/1: 172–179.

(4) Kohler, D., Böttcher, S., Krüger, M., Lehr, C.-M., Möhwald, H. & Wang, D. (2011): Template-assisted polyelectrolyte encapsulation of nanoparticles into dispersible, hierarchically nanostructured microfibers, Advanced Drug Delivery Reviews. 23: 1376–1379. doi:10.1002/adma.201004048.

(5) Murgia, X., Loretz, B., Hartwig, O., Hittinger, M. & Lehr, C. M. (2017): The role of mucus on drug transport and its potential to affect therapeutic outcomes. Adv Drug Deliv Rev. 2017 Oct 26. doi: 10.1016/j.addr.2017.10.009.

(6) Kuehn, A., Kletting, S., de Souza Carvalho-Wodarz, C., Repnik U., Griffiths, G., Fischer, U., Meese, E., Huwer, H., Wirth, D., May, T., Schneider-Daum, N. & Lehr, C.-M. (2016): Human alveolar epithelial cells expressing tight junctions to model the air-blood barrier. ALTEX 33/3: 251-260.

(7) Thews, G. & Vaupel, P. (2005): Vegetative Physiologie. Springer Verlag, Heidelberg.

(8) Cheers, G. (ed.) (2000): Anatomica. Global Book Publishing Pty. Ltd. Deutsche Ausgabe 2004. Tandem Verlag GmbH, Potsdam.


(10) Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts; K. & Walter, P.  (2011): Molekularbiologie der Zelle. 5. Auflage. Verlag Wiley Blackwell.

(11) Wennemuth, G. (2012): Taschenatlas Histologie. Urban & Fischer, München.

(12) Gollwitzer ES, Saglani, S, Trompette A, Yadava K, Sherburn R, McCoy KD, Nicod LP, Lloyd CM & Marsland BJ (2014):  Lung microbiota promotes tolerance to allergens in neonates DP-L1. Nat Med. 20 (6) :642-647. doi: 10.1038/nm.3568.

(13) Yun, Y, Srinivas, G, Kuenzel, S, Linnenbrink, M, Alnahas, S, Bruce, KD, Steinhoff, U, Bainesm JF & Schaible UE (2014): Environmentally Determined Differences in the Murine Lung Microbiota and Their Relation to Alveolar Architecture. PLOS ONE December 3, 2014. DOI: 10.1371/journal.pone.0113466

(14) Speckmann, E.-J., Hescheler, J. & Köhling, R. (2008): Physiologie. Urban & Fischer Verlag, München.

(15) European Respiratory Society (eds. 2017). European Lung White Book. Respiratory Health and Didease in Europe. Sheffield. Available at: [Accessed 26 Sep. 2017].

(16) Merkblatt zu der Berufskrankheit Nr. 1315 der Anlage zur Berufskrankheiten-Verordnung (BKV) "Erkrankungen durch Isocyanate, die zur Unterlassung aller Tätigkeiten gezwungen haben, die für die Entstehung, die Verschlimmerung oder das Wiederaufleben der Krankheit ursächlich waren oder sein können". Bek. des BMGHS, BarbBl. 3/2004, S.32.



(19) Schütt, C. & Bröker, B. (2011): Grundwissen Immunologie. Spektrum Verlag, Heidelberg.




(23) Aline Chary, Jennifer Hennen, Sebastian G. Klein, Tommaso Serchi, Arno C. Gutleb & Brunhilde Blömeke (2017): Respiratory sensitization: toxicological point of view on the available assays. Arch Toxicol (2017).

(24) Klein, S. G., Serchi, T., Hoffmann, L., Blömeke, B. & Gutleb, A. C. (2013): An improved 3D tetraculture system mimicking the cellular organisation at the alveolar barrier to study the potential toxic effects of particles on the lung. Particle and Fibre Toxicology 10: 31.


(26) Fitzpatrick, S. (2017):  ‘Organs-on-Chips’ Technology: FDA Testing Groundbreaking Science. Posted on April 11, 2017 by FDA Voice.


(28) Environment Directorate Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, Pesticides and Biotechnology (2016): Guidance Document on Considerations for Waiving or Bridging of Mammalian Acute Toxicity Tests. Series on Testing & Assessment Nr. 237. ENV/JM/MONO(2016)32.


(30) Belinsky, S. A., Stefanski, S. A. & Anderson, M. W. (1993): The A/J Mouse Lung as a Model for Developing New Chemointervention Strategies. CANCER RESEARCH 53: 410-416.



(33) Mas, C., Benainous, H., Huang, S., Wiszniewski, L. & Constant, S. (2017): Oncogenic Transformation of a Functional 3D Human Airway Epithelium for In Vitro Lung Cancer Modelling. American Journal of Respiratory and Critical Care Medicine 195: A2370.

(34) Skardal, A., Murphy, S. V., Devarasetty, M., Mead, I. et al. (2017): Multi-tissue interactions in an integrated three-tissue organ-on-a-chip platform. Scientific Reports 7: 8837, DOI:10.1038/s41598-017-08879-x





(a) a1-Antrypsin: a proteinase inhibitorthat contributes to an important enzyme (elastin) being enzymatically broken down in the alveoli in the lung, resultng in emphysema (35, 36).

(b) systemic: meaning arterial circulation, in the blood vessels.