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Executive Summary:
The present Final Report highlights the positive and extremely promising results of the LSE Project funded by the FP7 Programme. The experience Creabilis and its know-how on LSE modification of molecules was successfully combined with the expertise of the University of Dundee in the field of systems biology and network pharmacology.

Kinases are attractive targets in inflammatory and autoimmune diseases as they modulate the signalling pathways underlying these cellular responses and play a pivotal role in the initiation, propagation and regulation of immunologic responses
In contrast to applications in oncology, where kinase inhibitors represent the most popular class and efficacy is by far the most important selection criteria, the combination of efficacy and favourable safety profile is often key for the successful treatment of chronic inflammatory and/or autoimmune diseases with kinase inhibitors.
Creabilis is a specialized in the expert exploitation of the Low Systemic Exposure (LSE) approach, invented in the company over ten years ago. The technology has already been applied to kinase inhibitors and other bioactive molecules, giving the possibility to build a unique know-how in the field. The new chemical entities generated by the LSE approach have peculiar physico-chemical and pharmacological features, distinct from the parent molecule, yielding to molecules optimised for the topical routes of administration. Indeed, LSE-modified molecules show high local concentration with poor systemic absorption and negligible distribution in body tissues other than the target tissue, thus minimising the possibility of systemic toxicity. This would likely widen the range of patients affected by inflammatory pathologies that can benefit from topical kinase inhibition as a therapy and provide substrate for new medicines to be developed.
A careful computational database mining procedure allowed the identification of existing kinase inhibitors having a selectivity profile matching the kinase signalling misregulations involved in the three target therapeutic indications (Atopic Dermatitis, Psoriasis, Inflammatory Bowel Disease). Starting from the selected molecular scaffolds, a total of six LSE variants were synthesized and fully characterized. After in vitro activity screening, three molecules were chosen and progressed to in vivo testing on experimental models of Dermatitis and Psoriasis. No molecules passed the in vitro test stage for IBD. Two of the in vivo tested molecules evidenced very promising results that strongly recommend further developments.

It can therefore be concluded that each of the four main practical objectives of the LSE project, i.e. I) Identification of molecules for LSE modification; II) Synthesis of LSE molecule series; III) In vitro molecule selection; IV) In vivo molecule selection, has been met.
The top-level objective of the Project to design a better drug based on using a novel systems biology approach has also been met, allowing the generation of two innovative selective kinase inhibitors anticipating high local efficacy and a favourable safety profile in the treatment of Atopic Dermatitis and Psoriasis, respectively.
Project Context and Objectives:
Protein kinases regulate cellular signal transduction pathways: aberrant kinase activity and/or overactivity have been implicated in the aetiology of many human diseases. Kinase inhibition offers a powerful therapeutic strategy and kinases represent a drug target of major importance. Indeed, over the last decade, kinases have emerged as attractive therapeutic targets in inflammatory diseases. Numerous efforts in the pharmaceutical community are directed towards the discovery of small molecule inhibitors that regulate kinase function.
However, a number of issues need to be addressed when considering kinases as targets in inflammation, in particular selectivity and pharmacodynamics. These are mainly the selectivity of both the target and the inhibitor and the associated side effect profile of the corresponding drugs. There are over 500 protein kinases (the ‘kinome’) in the human genome. Large-scale screening of clinical kinase inhibitors across hundreds of kinases has revealed that many of the currently approved kinase drugs are highly promiscuous, binding to dozens of kinases. The combination of efficacy and favourable safety profile is often key for the successful treatment of chronic inflammatory and/or autoimmune diseases with kinase inhibitors. In order to get this, the LSE project proposed a novel drug discovery strategy that combines several innovative computational and experimental approaches to overcome challenges of selectivity and pharmacodynamic translation.
The strategy included two approaches:
i.) A computational approach based on network pharmacology to predict the best kinase targets and the associated chemical structures of known kinase inhibitors with the strongest disease association;
ii.) The modification of the selected compounds to produce new chemical entities by the so called LSE (Low Systemic Exposure) approach that generates innovative molecules with unique physico-chemical and pharmacological features
To achieve the first goal, the University of Dundee developed in silico methods for predicting the polypharmacology profile of compounds across hundreds of drugs. The network pharmacology approach aims to identify compounds with profiles that target a disease-specific spectrum of kinases.
The in silico network pharmacology method exploits a wide variety of biological data from kinase pathways in all relevant organisms to develop a novel and cost effective method to address medical and clinical needs.
With regards to the second strategic approach, the LSE proprietary technology conjugates small molecules to a low molecular weight amphiphilic polymer via a stable covalent bond. This gives rise to unique physico-chemical and pharmacological characteristics yielding molecules optimized for the different topical routes of administration. These molecules combine high local concentration with poor systemic absorption and distribution. If absorbed, LSE molecules are rapidly eliminated from the systemic circulation (very short plasma half-life), thus generating no or very low systemic side effects.

The LSE project had four main practical objectives:
I) Identification of molecules for LSE modification;
II) Synthesis of LSE molecule series;
III) In vitro molecule selection;
IV) In vivo molecule selection.

and a top-level objective, i.e. ‘to design at least an effective drug based on the latest robust science, safe and well tolerated with low systemic effects by using a novel systems biology approach and the LSE platform’. All objectives have been met

Project Results:
The tasks related to Work Package 1 (WP1) of the LSE Project consisted in the development of an overall network pharmacology and computational chemistry approach that led to the in silico identification of molecules suitable for LSE modification. This activity defined the optimum ‘target product profile’ expected of the final selected compounds for polymer conjugation. The criteria of the ‘target product profile’ contained the desired predicted drug properties for LSE modification and the optimum multi-kinase inhibition (polypharmacology) profile to treat the three immunological diseases of interest: atopic dermatitis (AD), psoriasis (PSO) and inflammatory bowel disease (IBD).
A computational network pharmacology approach was developed to systematically organize, integrate, and prioritize existing kinase pharmacology data to define kinase drug profiles that are most likely to modulate the above immunological diseases. This allowed to identify the optimum polypharmacology kinase profiles desired in the clinical candidate.
The criteria for the target product profile had previously been identified as follows:

- Multi-target kinase profiles for each indication
- Strong evidence of disease association for each target in the profile
- Known drug-like chemical matter in common
- Sufficient potency versus each target
- Evidence for selective modulation
- Amenable to LSE-modification
- Commercially available/readily synthesizable

It was developed a data-driven computational approach that leverages systematic text mining and large-scale public chemogenomics data to identify multi-target profiles that fulfil all of the above criteria. Importantly, the approach was completely generic and could easily be adapted to identify polypharmacology profiles for any disease indication.
The result was represented by a prioritized list of the most suitable kinase inhibitors per each indication, that represented the Objective O1 of the project, i.e. the identification of molecules for LSE modification, and the first milestone for all the indications (AD, PSO and IBD).
After the identification of a number of promising molecules through the in silico studies of WP1, the next step was the synthesis of LSE molecules for both in vitro and in vivo testing programmed in WP2. A preliminary study of several different synthetic pathways was performed together with the choice of the polymer type and chain length, the position of conjugation, the selection of the most suitable chemical bonds, and possible linkers and spacers between the active molecule and the polymer chain.
Once the candidate to conjugation and the most promising conditions were selected, a laboratory screening was performed in order to define the most appropriate reaction and purification conditions and a synthesis campaign was started to provide the amount of material (usually in the milligram scale) required for structural characterization and preliminary in vitro activity tests for hit compound selection.
Two LSE compounds were generated for each of the therapeutic indication: CT101 and CT102 for the treatment of AD, CT103 and CT104 for PSO, then CT105 and CT106 for IBD.
The quality of all these materials was evaluated by HPLC, NMR and mass spectrometry. allowing to obtain structural confirmation for all the six compounds and to meet Objective O2, i.e. the synthesis of LSE molecule series.
In order to identify the hit compound for each indication to be transferred to the scale up manufacturing step, biochemical and in vitro activity studies were conducted on selected molecules (WP3). CT102 was not progressed to this stage after a careful consideration of structure-activity relationship aspects that could potentially affect its pharmacodynamics.
The kinase inhibition profile for all the other five LSE molecules was determined by screening their inhibitory activity against the full panel of available human wild-type kinases (ca. 270). The percentage inhibition of each tested compound was calculated at a fixed concentration.
Unexpectedly, the two LSE compounds for the IBD indication (CT105 and CT106) showed an unfavourable kinase inhibition profile, suggesting not to proceed with further characterization. The decision was taken to address the remaining project resources to the evaluation of both CT103 and CT104 as hit candidates for the psoriasis indication.
The potencies of the remaining three LSE molecules (CT101, CT103 and CT104) were then measured on some of the most inhibited and interesting kinases properly selected for each compound, showing IC50 in the low nanomolar to low micromolar range.
CT101, CT103 and CT104 demonstrated to be active on cellular models related to their mechanisms of action and the target pathologies, thus confirming effective kinase inhibition in more complex biological systems and suggesting to progress all three LSE compounds to in vivo testing (Objective O3).
Furthermore, in order to study the interaction kinetics with their target kinases, Surface Plasmon resonance (SPR) testing was performed on all three conjugates. Despite relevant experimental problems affecting one of the programmed tests, the results obtained allowed the characterization of the drug dissociation kinetics from their target. The extracted relevant kinetic data, i.e. association rate (kon) and dissociation rate (koff) constants, and the calculated equilibrium binding affinity constant KD (KD=koff/kon) proved to behelpful in understanding the molecular implications of LSE.

Prior to enter the in vivo phase, the three hit compounds were progressed to the production scale-up stage. The structure of the selected compounds was analyzed with the aim of evaluating the theoretically possible synthetic routes that could represent favourable alternatives to the lab scale procedure. The feasible synthetic strategies were tested, and reaction conditions were optimized analyzing the effect of several variables (temperature, solvent, coupling reagent used) on main crucial aspects such as yield, product purity, and work-up feasibility. The outcomes of this activity were defined synthetic protocols optimized in terms of yield, purity profile and quality of the impurities, process robustness and applicability on a larger industrial scale.
A proper amount of Reference Material was also generated for each novel LSE molecule. The chemical stability was assessed by forced degradation procedures able to reproduce and enhance the potential effect of physico-chemical agents such as temperature, moisture and light exposure.
In the frame of WP4, as expected, the pharmacokinetic profiles of CT101, CT103 and CT104 showed the typical behaviour of all Creabilis’ topical-by-design LSE molecules, with low systemic absorption upon epicutaneous administration and fast systemic clearance coupled with negligible tissue distribution upon i.v. administration, thus suggesting the intrinsic safety profile typical of LSE molecules.
CT101 in vivo efficacy was assessed in well-established and validated experimental models of atopic and contact dermatitis. In both models, appropriate topical formulations of CT101 showed significant activity, reducing the typical hallmarks of both pathologies (e.g. skin swelling, erythema) without the side-effects of the topical corticosteroid used as positive control.
Of the remaining two LSE molecules selected for the psoriasis indication, only CT103 showed promising activity on the imiquimod animal model. CT103 succeeded in alleviating the symptoms of the target disease without the drawbacks of corticosteroid administration.
As requested by Objective O4, the results of the activities performed within WP4 allowed the selection of CT101 and CT103 as lead molecules for further pre-clinical and clinical development.

At the end of this 30 month project, two novel compounds were designed, synthesized, characterized and selected for future development to treat atopic dermatitis and psoriasis, respectively.
The generated lead compounds are expected to combine excellent safety profile with efficacy and optimised selectivity. This would tremendously widen the range of patients affected by inflammatory pathologies that could benefit of topical kinase inhibition as a therapy and provide an innovative concept for new medicines with unprecedented mechanisms of action to be developed.

Potential Impact:
The LSE project aim was to address current issues in the pharma industry regarding the effective treatment of important inflammatory disorders with an innovative drug design approach based on conjugating systems biology and network pharmacology methods to the so called Low Systemic Exposure technology (LSE). The selected three lead compounds are novel kinase inhibitor drugs anticipated to be effective, safe, and well tolerated with low systemic effects for the treatment of two important immunological skin diseases, i.e. atopic dermatitis and psoriasis.
The exploitation strategy is based on the development and selection of new lead compounds that are ready for entering the regulatory preclinical development stage, with a good chance to be commercialised in the medium/long term, both directly or via licensing. Creabilis will be pursuing four separate tracks to ensure that the FP7 project results are properly exploited: interactions with Key Opinion Leaders (that is vital to determine whether the FP7 LSE drugs will fill gaps in the market and address unmet patient need), a business development (BD) track (to identify the key pharmaceutical companies and BD contacts that may be interested in the potential drugs developed from the FP7), a financing track (to enable the development of the identified compounds, through source of potential future funding consisting of Venture Capital (VC), Angel, Institutional investors, etc.), and a future collaboration track (to seek collaborations required for the further development of these compounds to market).
Since the LSE molecules are not prodrugs releasing the active molecule where it is needed, but real new chemical entities, they can be covered by new intellectual property that will be patent protected. Indeed, the LSE technology generates novel compounds with unique physico-chemical and optimised pharmacological characteristics, thus resulting in the creation of new IP, which is crucial for the subsequent commercial exploitation of the results generated from the LSE project. Creabilis already has two patents in this regard from new molecular entities created from the LSE technology, and intends to adopt an analogue IPR strategy in parallel to the development of the lead compounds generated in this FP7 project.
Thanks to the extensive effort and time put in by all members of the consortium during the project, it can be concluded that project progresses were made towards proof-of-concept and exploitation in a clinical setting following the project conclusion. At the end of the project, the lead compounds have been identified, undergone LSE modification, and been tested in both in vitro and in vivo disease models. Following the project conclusion, an in-depth assessment will be made with regards to the experiments (both in vitro and in vivo) that may need to be done immediately post-project. However, most likely the next stage of drug development will involve all the Chemistry, Manufacturing and Controls (CMC) activities, as well as the full regulatory toxicology package to ensure a smooth transition towards clinical exploitation.
In terms of potential impact of the project results, the identified drug candidates are expected to be innovative topical selective kinase inhibitors directly addressing the medical need for new and effective anti-inflammatory drugs with local action and an excellent safety profile. This would tremendously widen the range of patients affected by inflammatory pathologies that could benefit of topical kinase inhibition as a therapy and provide an innovative concept for new medicines to be developed. From a methodological perspective, a key expected impact of the FP7 call, i.e. the development of innovative solutions in the area of systems biology for medical and clinical applications, was met. The approaches that were used, both in terms of systems biology through the development and application of new algorithms to identify kinase targets and relevant small molecule drug targets, and the LSE approach used to modify the identified drug targets, are indeed highly innovative.

No scientific publications have resulted from the LSE project so far. The main reason is the confidential nature of the project results, and the potential implications for future IP. Any inappropriate, untimely publication could significantly damage future attempts to industrially exploit the achievements of the LSE programme.
Of course, the consortium will be aiming at publishing the key results of the FP7 project in high quality, high impact, peer-reviewed international journals, but this will be delayed until a definitive decision about the protection has been made (through IPR or trade secrets), in agreement to the provisions of the Guide to Intellectual Property Rules for FP7 projects.

No application for patents, trademarks, registered designs, etc. has been made so far, since the most relevant project results came into light towards the very last period of the project itself.
However, Creabilis is fully aware that the IP protection is crucial, especially being an SME in the private sector. An active approach will thus be taken to IP management: all the generated compounds that are modified by the LSE approach are new chemical entities that can result in the generation of new IP. This new IP will be protected to ensure that the selected compounds can be properly exploited and developed.

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Contact details
Creabilis Therapeutics Srl
Via Ribes, 5
10010 Colleretto Giacosa (TO)

Phone +39 0125 53543