The value of human tissue studies in Translational Research

The increasing gap between R&D expenditures and the approval of new medicines by regulatory authorities is a matter of debate between R&D managers of the pharmaceutical industry since the 2000s.

An analysis of the number of NMEs (New Molecular Entities) approved by the US Food and Drug Administration (FDA), leads to the conclusion that most of the top pharmaceutical companies did not launch enough new drugs in the past years to achieve the reported 2-3 NMEs/year/company which would be necessary to meet their growth objectives (1). Despite huge human resources and financial investments, the Pharmaceutical Research and Manufacturers of America reported that for every 5,000 to 10,000 compounds that enter the pipeline, only 1 receives approval (2).

The total worldwide R&D spend of pharmaceutical and biotechnology companies increased from $108bn in 2006 to $141bn in 2015 (3). The use of new technologies supposed to reduce the timelines in drug discovery, such as combinatorial chemistry, DNA sequencing, high-throughput-screening (HTS) or computational drug design, have instead increased dramatically R&D costs. Since the estimated costs for drug discovery and preclinical development accounts for 33% of the total expenditures to put a New Chemical Entity (NCE) on the market (4), let’s take a look at the possible reasons for the counter-performance of drug discovery process.

What is going wrong in the process of preclinical studies?

Multiple reasons have been reported to explain the failure of drug development programs lately. Although adverse effects, toxicity, or pharmacokinetic features are frequently cited as reasons for arrested drug development, a review of the portfolio performance of a Big Pharma (5) suggests failures in Phase II studies are mainly caused by a lack of efficacy.

This fact raises the question on the translational values of preclinical tests used to select and assess the efficacy of drug candidates. In our opinion, one explanation for the lack of efficacy in clinical trials could be that too many drugs proceed to clinical trials with minimum human data. Unanticipated species differences may lead to clinical observations in humans that were not detected in preclinical animal species and vice versa.

As a matter of fact, the translational value of widespread in vitro assays was recently questioned. Cell-based assays lose functional relevance and do not retain physiological cell-to-cell relationships in a 3-D structure. Reconstructed or engineered 3-D tissues produced from stem cells or as reconstructed organoids fail to reflect the actual disease phenotype and diversity of responses found in healthy and diseased tissues obtained directly from patients.

It is also important to be aware of important anatomical differences between experimental animals and humans. Comparing internal structures of vital organs like heart, lungs and brain is essential to understand human physiology and to offer innovative treatments for chronic or degenerative diseases. A striking example comes from the recent discovery of “rosehip neurons”, a new type of inhibitory neuron present in human, but not mouse brains (6).

Ongoing efforts to inventory all the cell types in the human brain may well turn up similar discoveries in the future, suggesting this type of finding could be the tip of the iceberg, not only in the brain, but also in many other organs. In another striking example published last month, two independent research teams (7,8) used single-cell RNA sequencing to generate detailed molecular atlases of mouse and human airway cells. Their work revealed the existence of a previously unknown cell type (ionocytes) that expresses high levels of the gene mutated in cystic fibrosis, the cystic fibrosis transmembrane conductance regulator (CFTR). According to one of the authors, this discovery rewrites the way we think about lung biology and lung cells. However, the importance of this finding would have been underestimated without a direct comparison between mouse and human cells.

The advantages of using human tissues to study efficacy of drug candidates

Physiological, pharmacological and histological studies on fresh, frozen or Formalin-Fixed Paraffin Embedded (FFPE) human tissues are potentially useful for different purposes:

  1. A proof of concept to confirm efficacy data obtained previously in recombinant cells/animals.
  2. To discover potential side effects in vital organs like heart, lungs and brain that could be due to off-targets activities, specific to human being.
  3. To validate new therapeutic targets using biomarkers.
  4. To replace / reduce animal experimentation.

We, in Humana Biosciences think that human isolated tissues can be useful to test efficacy along with safety of drug candidates.

Functional studies on fresh isolated tissues can be used to predict clinical efficacy by understanding the behavior of the human drug target in its physiological environment. These assays are potentially useful to bridge the gaps between in vitro cell-based studies, in vivo animal studies and clinical trials. For example, using the organ bath technique to evaluate myogenic and neurogenic contractions/relaxations on fresh human tissues provides preclinical data that are more variable than similar studies performed on laboratory animal tissues but have the advantage to reflect the diverse patient population, so increasing test predictivity.

Moreover, the use of human specimen is more and more necessary in the era of personalized medicines; as the search for blockbusters decreases, the need for targeted therapies based on predictive data obtained in  sub-populations of patients will certainly increase in the next few years.


There are several examples in the literature showing stunning differences in the nature of the receptor subtype involved in a chosen pharmacological effect of a neurotransmitter. On human isolated urinary bladder, it was reported that the potentiation of cholinergic neurotransmission by low concentrations of 5-HT (5-hydroxytryptamine)is mediated by 5-HT4 and 5-HT7 receptors (9). However, animal studies reported strong evidence for the involvement of different 5-HT receptor subtypes, e.g. 5-HT2 in dogs, 5-HT3 in rabbits and both 5-HT2 and 5-HT7 in rats (10).

Surprisingly, in the isolated urinary bladder of the Rhesus and Cynomolgus monkey, 5-HT4 receptors are involved in the opposite effects, e.g. an inhibition of cholinergic neurotransmission. So, studies on different animal species were not useful to understand the effect of 5-HT on the human urinary bladder and to characterize the receptors mediating the effects of this neurotransmitter.

Another example comes from a paper reporting an extensive characterization of prostaglandin receptors subtypes in human urinary bladder, showing evidences that receptors involved in contractile effects are completely different from those operating in rats (11). These differences may have contributed to the failure of an EP1 receptor antagonist for the treatment of Overactive Bladder in a recent clinical trial (12). Judging from published data, this drug candidate was extensively characterized in rats and other experimental animals, but its effects on human urinary bladder were probably not studied.

Sir James Black, Nobel Prize for Medicine in 1988 for his work leading to the development of propranolol and cimetidine, stated in a paper (13) published in 2010: “The choice of level for studying the pharmacology of complex systems in the first instance is the intact tissue bioassay. The attractiveness of these bioassays is that they can be driven chemically and physically in as many ways as our imaginations can conceive and yet still remain, potentially at least, mathematically tractable and analysable.” Unfortunately, the technological revolution of the last 20 years led to a rejection of functional assays by R&D departments of the pharmaceutical industry. Indeed, they were perceived as “old-fashioned pharmacology” which has resulted, in our opinion, in a great increase of ineffective or unsafe clinical candidates.

The advantages of using human tissues to study safety of drug candidates

Human tissue studies are also of value for non-clinical safety pharmacology studies in support of ICHS7A and ICHS6 guidelines for pharmaceuticals and biotechnology products, respectively. As an example, knowledge of how drugs affect the gastrointestinal (GI) tract is extremely important in the drug development process since studies have shown that 18% of all clinical adverse drug reactions are related to adverse GI effects (14). A wide variety of drugs currently on the market showed adverse effects on GI function,with more than 700 drugs shown to cause diarrhoea (15). Unfortunately, adverse effects seen in preclinical toxicology species may not be representative of the human condition. An understanding of a compound’s impact on the human GI tract prior to clinical trials is valuable for making informed decisions on the progress of a drug development candidate.

Gut motility effects can easily be assessed in organ bath experiments using muscle strips. Strips can be stimulated with electrical impulses or drugs and the effects of test compounds on these responses evaluated. As an example, on longitudinal muscles from human colon, repetitive electrical field stimulations caused enteric nerve stimulation resulting in sustained contractions.  Addition of the neurotransmitter 5-hydroxytryptamine (5-HT) caused a potentiation of the EFS-induced contraction, in this case by activation of the 5-HT2B receptor (16).

Information generated by human tissues may even be included in a clinical trial application or marketing authorization application. For example, the FDA review of gastrointestinal drugs used studies in fresh human coronary arteries to evaluate the safety of 5-HT4 agonists (17). This class of drugs that was found in the past to induce fatal cardiac arrhythmias and cardiovascular ischemic events followed by heart attack and stroke, leading to market withdrawal of Cisapride and Tegaserod (18).

In the UK, the National Center for Replacement, Refinement and Reduction of Animals in Research (NC3Rs) together with the Medicines and Healthcare Products Regulatory Agency (MHRA) are working to increase the uptake of human tissue-based approaches not only with the aim to replace the use of animals but also to provide more relevant and predictive tools to determine the safety of drugs entering clinical studies.



Despite these strong evidences of their usefulness, the use of human tissues in preclinical R&D labs of the pharmaceutical industry is still quite limited. The problem is not one of tissue availability; at least 600,000 surgical residual human tissues are generated annually in the UK and over 95% of patients are happy to donate their tissues to medical research, even to private companies (19). The underuse of human tissues is probably due to a combination of different factors: logistic difficulties in collecting tissue in time, lack of incentive on the part of surgeons and healthcare staff to carry out additional duties during surgery, the need for flexible working timetables by the staff testing fresh tissues and finally by technical difficulties.

Functional studies are almost always carried out in fresh tissue samples. This type of study is technically challenging and with current preservation techniques, experiments must be carried out not later than 24 hours post-surgery. Considering now fresh-frozen or Formalin-Fixed Paraffin Embedded (FFPE) tissues, the relevance of the tissue is dependent on the quality of the collection, storage and preparation of the tissue prior to experiments. Extensive characterization of the tissue is often carried out prior to or during experiments to ensure reliable results. For example, a RIN score (RNA Integrity Number) of 7.0 or higher is appropriate to ensure the quality of RNA isolated prior to any investigation into gene expression levels or biomarkers characterization in healthy and diseased tissue samples.

In conclusion, only specialized, flexible and reactive CROs, having years of experience in human tissue research, are able to overcome the complexity of this type of preclinical work.

There is clearly sufficient fresh tissue and public support to allow its use as an essential and routine element of drug development; however, a greater cooperation between public hospitals, bio-banks, regulatory bodies, preclinical CROs and pharmaceutical companies is needed to support and implement the use of fresh human tissues for preclinical research studies, particularly for a final validation of drug candidates.


  1. Munos B. Lessons from 60 years of pharmaceutical innovation. Nat Rev Drug Discov. 2009;8:959–68.
  1. Pharmaceutical Research and Manufacturers of America: 2013 Pharmaceutical Research Industry Profile. Published April 2013, Page 32.
  2. 3. Evaluate Pharma. World Preview 2015. 2015. ONLINE 13 Sept. 2018
  1. Paul SM, et al. How to improve R&D productivity: the pharmaceutical industry’s grand challenge. Nat Rev Drug Discov. 2010;9:203–14.
  1. Cook D. et al., Nat Rev Drug Discov. 2014; 13:419–431
  2. Boldog, E.; Bakken, T..; Hodge, R.D..; Novotny, M. Aevermann, B.D. Baka, J.; Bordé, S.; Close, J. L., Diez-Fuertes, F. Nature Neuroscience 2018, 21 (9): 1185–1195.
  3. D.T. Montoro et al., “A revised airway epithelial hierarchy includes CFTR-expressing ionocytes,” Nature. 2018; 560(7718):319-324.
  4. L.W. Plasschaert et al., “A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte,” Nature, doi:10.1038/s41586-018-0394-6, 201
  5. D’Agostino G, Condino AM, Gallinari P, Franceschetti GP, Tonini M. . J Pharmacol Exp Ther. 2006, 316(1):129-35.
  1. Palea S, Lluel P, Barras M, Duquenne C, Galzin AM, Arbilla S. Involvement of 5-hydroxytryptamine (HT)7 receptors in the 5-HT excitatory effects on the rat urinary bladder. BJU Int. 2004, 94:1125-31.
  2. Root JA, Davey DA, Af Forselles KJ. Prostanoid receptors mediating contraction in rat, macaque and human bladder smooth muscle in vitro. Eur J Pharmacol. 2015, 769:274-9.
  3. Chapple CR, Abrams P, Andersson KE, Radziszewski P, Masuda T, Small M, Kuwayama T, Deacon S. Phase II study on the efficacy and safety of the EP1 re-ceptor antagonist ONO-8539 for non-neurogenic overactive bladder syndrome.Urol., 2014, 19:253–260.
  4. Black J. Reflections on drug research. Br.J. Pharmacol., 2010; 161: 1204–1216
  5. Keating C, et al. 2010. The validation of an in vitro colonic motility assay as a biomarker for gastrointestinal adverse drug reactions. Toxicology and Applied Pharmacology, 245, 299.
  6. Chassany O, et al. 2000. Drug-induced diarrhoea. Drug Saf, 22, 53.
  7. Borman R, A, et al. 2002. 5-HT2B receptors play a key role in mediating the excitatory effects of 5-HT in human colon in vitro. Br. J. Pharmacol, 135, 1144
  1. FDA Centre for Drug Evaluation and Research, Gastrointestinal Drugs Advisory Committee, 17 November 2011, p. 95 [online].
  1. Sahu RK, Yadav R, Prasad P, Roy A and Chandrakar S. Adverse drug reactions monitoring: prospects and impending challenges for pharmacovigilance. SpringerPlus 2014, 3:695

Dr. Stefano Palea, CEO Humana Biosciences

Author information

Dr. Stefano Palea has 30 years of experience in preclinical Research, in both big pharma (GSK, Sanofi) and Preclinical CROs.

Key achievements

  • Cofounder and CSO of UROsphere SAS (2004-2014), a preclinical CRO, spin-off from Sanofi-Aventis
  • Contributed to UROsphere annual turnover growth from 0 to 1 million € (2005-2008)
  • Head of the scientific team generating experimental data for a new patent for an old drug (litoxetine, IXA-001) previously developed by Sanofi-Aventis (Drug Repurposing)
  • Head of the team responsible for IXA-001 patent awarding in EU, Canada, USA and Japan
  • Cofounder of IXALTIS SAS, a specialty pharmaceutical company developing proprietary therapeutics to treat urinary incontinence in women and men. On May 2016, IXALTIS successfully completed its first fundraising round, securing 8 million € to perform Phase II studies with IXA-001 in EU and USA.
  • Coaching and Business Development for 3 startups operating as preclinical CROs in France (BioTechBank, E-Phys, Alphenyx

Dr. Palea is the author of 40 peer-reviewed scientific papers and more than 50 oral presentations and posters at international meetings.


For further information

Dr. Stefano Palea

HUMANA Biosciences SAS
Prologue Biotech
516, rue Pierre et Marie Curie
31670 Labège, FRANCE