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Preclinical development of drugs and drug delivery technology for the treatment of inherited photoreceptor degeneration

Final Report Summary - DRUGSFORD (Preclinical development of drugs and drug delivery technology for the treatment of inherited photoreceptor degeneration)

Executive Summary:
Hereditary retinal degeneration (RD) relates to a group of currently untreatable neurodegenerative diseases affecting photoreceptors, leading to blindness. The overall goal of the DRUGSFORD project was to develop compounds (drugs) and delivery systems for the treatment of RD. Dysregulation of cyclic guanosine monophosphate (cGMP) is a pathological hallmark of RD and cGMP-signalling was therefore chosen as the target for the development of new pharmacological approaches to treat RD.
The DRUGSFORD project has generated a large number of new cGMP analogues (cyclic nucleotides) to efficiently bind and inhibit the cGMP targets, i.e. protein kinase G (PKG) and the cyclic nucleotide gated channel (CNGC). Cyclic nucleotides were combined with a liposomal drug delivery system to mediate transfer of compounds across the blood retinal barrier and transfer the compounds to the photoreceptors of the retina.
Notable project developments include the identification of the lead compound DF003 and its liposomal formulation LP-DF003, which showed protective properties in several in vivo RD models. Using a variety of in vitro techniques LP-DF003 was shown to efficiently inhibit both PKG and CNGC at low micromolar concentrations. LP-DF003 significantly increased photoreceptor viability in the rd1, rd2, rd10, and cpfl1 animal models for RD. Increased viability was accompanied by a significant and marked improvement of in vivo retinal function. Preliminary in vitro and in vivo toxicity assessments revealed no major adverse effects of DF003 or LP-DF003, even at concentrations of up to 500 µM. Likewise, further in silico toxicity simulations (DEREK Nexus, Leadscope) revealed an expected, class-typical possibility for clastogenicity, but did not indicate any major organ toxicity of the DF003 compound. A subsequent in vivo safety and tolerability study in non-human primates (NHP), using concentrations of up to 100-fold of the estimated human therapeutic dose, indicated no major DF003 or LP-DF003 related adverse effects. In parallel to the pharmacological experiments, a new chemical synthesis process allowing efficient production scale-up according to the standards of good manufacturing practice (GMP) was developed for DF003.
A first patent for the LP-DF003 compound formulation was filed on 15th March, 2015 (PCT/EP2016/055659) with further patent applications on novel cyclic nucleotides submitted on 11th July, 2016 (EP16178924.3) and 31st August, 2016 (EP16186700.7). Furthermore, the lead compound DF003 also obtained an orphan drug designation (ODD) from the European Medicines Agency (EMA) on 18th March, 2015 (EU/3/15/1462).
Overall, during the four project years, DRUGSFORD has achieved all major goals and milestones defined at project start. DRUGSFORD demonstrated a strong photoreceptor protective potential of inhibitory cGMP analogues and that liposomal encapsulation achieves efficient drug targeting to the photoreceptors of the retina. The latter data can be important for development of new therapies for the retina in general. Importantly, the LP-DF003 pharmacological treatment markedly preserved in vivo retinal function and counteracted photoreceptor degeneration in four different in vivo RD models. With these cGMP analogues DRUGSFORD has introduced a new class of compounds for RD treatment, inventively combined with an efficient retinal delivery method. Importantly, at project conclusion, with the establishment of a manufacturing process and the successful preclinical efficacy as well as safety and tolerability testing, the lead compound formulation LP-DF003 is in principle ready to enter clinical trials. This provides a clear perspective for a clinical translation of DRUGSFORD results in the near term and could bring the very first pharmacological treatment to patients suffering from RD.

Project Context and Objectives:
The retina is a neuronal tissue that sits at the back of the eye, and the retinal photoreceptors – rods and cones – are the primary neurosensory cells with which we perceive light. Whereas in healthy individuals the photoreceptors usually function well for many decades, people carrying certain gene mutations experience photoreceptor loss, severe visual handicap eventually leading to blindness. Loss of photoreceptors is the hallmark of a genetically and clinically heterogeneous set of rare diseases commonly referred to as hereditary retinal degeneration (RD). Although many of the causative mutations have been defined, to date RD is still untreatable and the subsequent degeneration mechanisms are still poorly understood. In many cases, RD mutations lead to an excessive accumulation of cGMP in photoreceptors (Arango-Gonzalez et al. 2014). Increased cGMP can be envisaged to have two main targets: 1) cGMP dependent protein kinase (protein kinase G; PKG), which when activated by cGMP, will phosphorylate specific proteins, and 2) cyclic nucleotide gated ion channels (CNGC), which allow for a cGMP controlled influx of Na+ and Ca2+. Both cGMP targets are connected with photoreceptor degeneration (Paquet-Durand et al. 2009; Paquet-Durand et al. 2011) and can thus be regarded as disease drivers (Trifunović et al. 2012).
cGMP-signalling is central in phototransduction, although excessive cGMP production or insufficient hydrolysis may trigger photoreceptor degeneration. The actions of cGMP and its targets can be rebalanced by cGMP analogues. The DRUGSFORD partner BIOLOG had unique experience and know-how in the chemistry of nucleotide analogues, and had previously developed highly specific compounds affecting PKG and CNGC activity. BIOLOG had already discovered a structural basis for inhibiting CNGC and PKG, and could use this expertise to generate novel structures with increased lipophilicity, and high resistance towards hydrolysis.
Drugs for retinal diseases may be applied via a variety of different routes, including systemic intraperitoneal or intravenous injections, or directly to the eye by intravitreal or subtenon injections. Regardless of the application paradigm, any RD treatment must reach sufficient and sustained drug concentrations within photoreceptor cells, and to do this the compounds have to be effectively delivered to the retinal neurons across the blood-retinal-barrier (BRB). The BRB is an equivalent to the blood-brain-barrier (BBB) in other parts of the central nervous system, and is formed by blood vessel endothelial cells together with Müller glial cells. This leads to a separation of retinal neurons from the blood stream and the BRB hence acts as a major obstacle for drugs that target photoreceptors. To overcome this problem a drug delivery system (DDS) is needed, which, considering the lifelong treatment required in RD, should also allow for a safe and prolonged release of the drug to avoid frequent reapplications.
Photoreceptors are highly specialised for photon capture and transformation of stimuli into electrochemical signals. They display distinctive features, including the phototransduction signalling cascades and the architecture of transductive compartments and synapses, making photoreceptors notoriously difficult to study. The academic DRUGSFORD partners had a profound knowledge on photoreceptor physiology, degeneration, and experimental neuroprotective therapies. The experimental systems used were: (1) a cell-based system, in which photoreceptor-like cells were generated from retinal stem cells derived from wildtype or disease models; (2) an organotypic retinal explant culturing system, which, under serum free conditions, conveniently monitored the effects of various treatments on disease models; (3) in vivo systems using rodent animal models that allowed to fully exploit the unique diagnostic possibilities of the eye, i.e. direct, non-invasive visualisation and functional characterisation of neurodegenerative processes and treatment effects.

The three academic groups performed a step-wise testing of drugs and drug/DDS combinations for their photoreceptor neuroprotective properties, using these systems of increasing complexity to filter and select the best candidates for further development.
The general objectives for the DRUGSFORD project were thus defined as follows:
1) To develop and refine cyclic nucleotide based drugs targeting PKG, CNGC.
2) To develop and refine systems suitable for delivering these drugs across the BRB to photoreceptors and providing for sustained drug release.
3) To test and select drugs and DDS in experimental settings of increasing complexity, thereby completing all major stages of pre-clinical pharmacology testing.
4) To develop methods/assays for production of optimal drug/DDS combinations and complete toxicological tests required for entering clinical trials.

In addition to these objectives, the valorisation of research outcomes via patenting, scientific publishing, and possible commercialisation was to be conducted. To facilitate the final evaluation of the DRUGSFORD project, a number of performance and research indicators were defined at project start:
1) The generation of new compounds and their efficacy to inhibit their primary targets CNGC and PKG.
2) The generation of new drug/DDS combinations and their efficacy to deliver compounds to the photoreceptors, with therapeutic effects.
3) Obtaining patents for the above mentioned individual compounds and drug/DDS combinations.
4) Scientific publications testing and highlighting the properties of new compounds and DDS.
5) Further dissemination of DRUGSFORD project results on conferences and workshops.
6) Positive evaluation of one or more drug/DDS combinations in WP4, successful completion of GMP-like production and pre-clinical toxicology testing.

As laid out in more details in the “Main science & technology results/foregrounds” section and furthermore in the DRUGSFORD 3rd periodic report, at project conclusion DRUGSFORD has essentially reached all of its main objectives and satisfied all of the evaluation criteria mentioned above:
A large number of new cGMP analogous compounds were generated and shown to be highly effective inhibitors of CNGC and PKG. New drug/DDS combinations were shown to efficiently deliver compounds to the photoreceptors, with marked morphological and functional benefits in a variety of in vivo RD animal models. In total three new patent applications for compounds and drug/DDS combinations were filed and, in addition, an orphan drug designation (ODD) was obtained for the lead compound formulation LP-DF003. At present, more than 10 peer-reviewed scientific articles have been published – with several more to be published in the near future – and DRUGSFORD results have been presented via press releases, RD patient workshops, as well as on a number of international conferences. Finally, for the lead compound formulation LP-DF003, a new GMP-ready production process was established and pre-clinical safety and tolerability studies concluded with no major adverse findings.

Project Results:
Efficient drugs for RD treatment are regretfully still lacking. To address this urgent need, the DRUGSFORD project has generated a large number of new nucleotide based compounds to efficiently bind and interfere with the two pharmacological targets, namely protein kinase G (PKG) and the cyclic nucleotide gated channel (CNGC). The compounds were tested in a step-wise screening system, the complexity of which rose with each consecutive step, from cell free assays, to individual photoreceptor-like cells, to whole retinal tissues, to live animals. The positively tested compounds were combined with an innovative liposomal drug delivery technology to allow them to reach the neuroretina and the photoreceptors.
The main science and technology outcomes of the DRUGSFORD project are highlighted in the following and grouped along the lines defined by the work packages (WPs) in which they were achieved. Each of the six WPs had specialised tasks for all or several of the individual participants, which allows a highly parallelised work execution that eliminates unnecessary waiting times.

WP1. Successful development of novel drugs targeting the cGMP signalling cascade
In the beginning of the project a synthesis strategy was established that provided first for the generation of cGMP analogues that will efficiently activate PKG. Such compounds can be produced more easily than PKG inhibitors but can later – once their PKG activating effect is confirmed in cellular assays – be converted into inhibitory compounds. In total, 227 novel compounds were prepared, by the DRUGSFORD SME partner BIOLOG while at the same time the experimental basis for the successful encapsulation of these compounds into liposomes was established by the SME partner to-BBB. Pre-screening in a cell-free assay system identified a number of novel compounds with increased potency, including several dimeric cGMP analogues. Altogether 30 PKG activators were tested in a cellular test system developed by UNIMORE, the 661W cell line derived from murine retinal photoreceptor precursors. The most potent activators identified in the cellular testing were selected for conversion into the corresponding inhibitory compound. The generation of dimeric cGMP analogues yielded a number of novel compounds with increased potency. To efficiently test these novel inhibitory compounds, a new genetically modified cell line was developed. This 661W-NRL cell line mimics certain aspects of photoreceptor physiology and is suitable for medium to high-throughput drug screening. At a later stage and for further in-depth testing of novel compounds, photoreceptor-like cells were used that were differentiated from stem cells isolated from the ciliary margin of eyes from either wild-type (wt) or rd1 mutant animals. WP1 constituted the first stage in step-wise testing of novel compounds in three different experimental test systems of rising complexity (cell cultures -> organ cultures -> in vivo). In total 20 inhibitory compounds were tested in cell-based assays. The most successful inhibitory and activatory compounds were forwarded to in vitro testing in organotypic retina explant cultures and eventually further in vitro and in vivo validation in WP3.

WP2. Development of a system for in vivo drug delivery to retinal photoreceptors
The liposomal drug delivery system (DDS) originally developed by the company to-BBB was adapted to and tested for retinal drug delivery, the protocol for DDS formulation for cGMP analogues was established, while at the same time analytical methods for DDS detection were developed. The biocompatibility of the DDS (empty and fluorescein loaded liposomes) was tested in vitro and in vivo and importantly no evidence for toxicity was found. Using fluorescein loaded liposomes the successful delivery to retinal photoreceptors was demonstrated in vitro in cells. Fluorescein loaded liposomes were then used in five different in vivo application paradigms on wild-type mice and rats, namely topical application to eye, subtenon and intravitreal injection, as well as intravenous and intraperitoneal injection. Initially a combination of intravenous and intraperitoneal injection was established as the most suitable in vivo application paradigm. Later on the use of intraperitoneal injection alone was established as suitable as well and, because of its ease of use in animal experiments, was used for later drug testing in WP3 and WP4. This project part was successfully concluded at project month 18 already (cf. 18 months report).

WP3. Identification of a novel photoreceptor neuroprotective drug/DDS combination
A set of bioanalytical methods were developed for the detection of novel cGMP analogues in blood and tissue samples. A pharmacokinetic study revealed a strong extension of in vivo half-life and bioavailability of cyclic nucleotides in the blood stream when applied as liposomal formulation. This WP proceeded with the step-wise testing of novel compounds started in WP1 (cell cultures -> organ cultures -> in vivo). While in total twenty inhibitory compounds were tested in cell based assays at UNIMORE, fourteen of these compounds were tested on organotypic retinal explant cultures at ULUND, and finally the five most successful compounds were tested in vivo on rd1 mutant animals at EKUT. Importantly, for the best compound tested so far (DF003), both in vitro and in vivo, the liposomal formulation was more effective when compared to the naked, non-liposomal compound. The liposomal formulation of DF003 was therefore defined as the first lead compound, which underwent subsequent cross-model testing in several other animal models for RD (see WP4).

WP4. Confirmation and validation of marked morphological and functional rescue
RD is a genetically very heterogeneous disease with causative mutations in over 150 different genes. To ensure the successful commercialisation of a new treatment that will benefit as many RD patients as possible, it is therefore important to assess whether this new treatment can be beneficial in forms of RD caused by mutations in different genes. Consequently, the neuroprotective capacities of LP-DF003 tested positively in the rd1 mouse (WP3) were assessed in four genetically distinct RD animal models, including the rd2, rd10, and cpfl1 mouse, as well as the P23H rat. Importantly, systemic treatment with LP-DF003 via intraperitoneal injections showed clear morphological rescue effects in rd1, rd2, rd10 and cpfl1 animals, with additional significant and marked functional benefits observed in the rd2 and rd10 mouse, where such improvements can be readily analysed. In line with the results obtained in mice, there was a clear trend towards protection in the P23H rat, although these effects were statistically not significant. Consequently, LP-DF003 was selected as compound for manufacturing process development and initial regulatory preclinical testing in WP5.

WP5. Successful establishment of manufacturing process and regulatory preclinical testing
Because of the bankruptcy of the SME partner to-BBB, this WP experienced significant delays, which prompted the consortium partners to ask for a project extension and the inclusion of the new partner SPPD that would take over the remaining tasks of the former to-BBB. The extension and amendment of the DRUGSFORD project led to the implementation of a number of changes in the research programme, in order to take full advantage of the GMP-production and drug development capabilities of the new partner SPPD. SPPD developed a new chemical synthesis procedure for the production of DF003, which was 1) more cost-efficient than the original BIOLOG process, 2) amenable to scale-up to kg quantities, and 3) amenable for GMP-grade production. SPPD also undertook an initial in silico toxicity screen with the help of the CRO Toxicology Knowledge Team (TKT; Sweden). With help and guidance from SPPD, BIOLOG then performed a safety and tolerability study for DF003 and LP-DF003 in non-human primates (NHP) at the CRO Covance (Germany). This NHP study provided an initial safety and tolerability assessment for systemic and local application of both the “free” compound DF003 and the liposomal formulation LP-DF003. Importantly, this study revealed no major adverse effects of DF003 or LP-DF003 treatment in NHP at concentrations of up to 100-fold the expected human therapeutic dose.

WP6. Fruitful and effective project management
While this does not directly relate to science and technology developments, the successful project implementation strongly facilitated the latter and may even continue to do so in the future. Key reasons for the success of the DRUGSFORD may have been an effective project management, together with a strong focus on continued and very frequent communication among the unfailingly dedicated partners. To facilitate exchange of results and to allow for stringent planning and execution of project tasks, the project partners have met in consortium meetings every six months and throughout the project have had regular Skype video conferences (sometimes as often as twice per week). In addition, a final consortium meeting was organised in August 2016, in Tübingen, which summarised the latest project results and led to the starting of several follow-up initiatives. These include the possible founding of a company concerned with the further commercial development of DRUGSFORD project results, the planning for phase I/II clinical trial for the lead compound LP-DF003, including an EU grant application (Proposal ID: 754625-1; submitted 04/10/16), and the setting up of an international training network for PhD students.

Taken together, during four project years the DRUGSFORD consortium met all major project objectives, namely the identification of a drug/DDS combination that can effectively prevent photoreceptor death neurodegeneration in a variety of in vitro and in vivo models for hereditary retinal degeneration. Even the bankruptcy of partner to-BBB in project month 29 did not prevent the further development of the first lead compound LP-DF003 in terms of GMP-like production and manufacturing, as well as toxicological assessments. At this point, at project conclusion, the lead compound formulation LP-DF003 is essentially ready for clinical testing and a number of further novel compounds with strong biological activities have been identified and may be further developed as 2nd generation compounds.

Potential Impact:
Vision loss due to hereditary retinal degeneration (RD) is a devastating handicap with far-reaching effects on both the affected individuals´ quality-of-life and the society as a whole. Since RD is to date still without therapy, the overall goal of the DRUGSFORD project was to develop new compounds (drugs) and drug delivery systems (DDS) for its treatment. Many forms of RD are associated with pathologic changes in cGMP regulated events. Therefore, the DRUGSFORD consortium focussed on producing cGMP analogous compounds that can intervene with and rectify aberrant cGMP signalling. To this end the highly cGMP specific enzymes protein kinase G (PKG) and cyclic nucleotide gated channels (CNGC) were chosen as targets for novel compound development.

New compounds and technology for efficient drug delivery to the retina
At project conclusion, the combined efforts of the consortium have created benefits that reach not only the DRUGSFORD project as such, but also the individual partners. The many (more than 220) novel compounds generated will enlarge the product portfolio of the company BIOLOG and provide new possibilities for commercialisation. The adaptation and optimisation of the drug delivery technology of the company to-BBB to be used for cGMP analogues and retinal drug delivery expanded their technology to a new class of compounds and to applications in the retina. Although the SME to-BBB went bankrupt in project month 29, its intellectual property (IP) has been salvaged by a new company (2-BBB medicines), with new management, and 2-BBB medicines is expected to continue an eye related product line. The academic partners in the DRUGSFORD consortium have gained ample new insights into cGMP-signalling in photoreceptors and the retina as a whole, as well as developed new tools and acquired expertise in pre-clinical drug testing.

Novel neuroprotective drug formulation and intellectual property
From the outset, the ambition of the DRUGSFORD consortium was to have a first lead compound ready for clinical testing by the conclusion of the project. Very promisingly, the lead drug/DDS formulation LP-DF003 has tested positively in at least four different animal models for retinal degeneration, without any obvious signs for toxicity at therapeutic dosing. Further safety and tolerability testing, including in non-human primates did not reveal any major adverse events, highlighting the feasibility of an application in human patients Accordingly, the consortium has jointly filed for a patent for this compound (EP15159285.4) and in addition, successfully obtained an orphan drug designation for the use of DF003 for the treatment of RD from the European Medicines Agency (EU/3/15/1462). Two more patent applications (EP16178924.3 and EP16186700.7) for novel cyclic nucleotide compounds have been filed and will benefit and expand the product portfolio of the company BIOLOG.

Foundation of a new company dedicated to commercialisation of DRUGSFORD results
Taking advantage of the high momentum of the project, the DRUGSFORD partners together have agreed to found a joint company (provisional name “MIRECA medicines”; to be established in Tübingen, Germany). This new company will collect the common DRUGSFORD intellectual property and forward the lead compound formulation LP-DF003 to clinical testing and potentially commercialisation as the (to date) very first pharmacological treatment for RD. The company will also be devoted to extending the potential disease indications, in particular concerning rare diseases of the retina.

Moreover, second generation DRUGSFORD compounds and application-specific compound/DDS combinations will be further developed and investigated in parallel. Importantly, the experiences and know-how gained in the pre-clinical development of the first lead compound DF003 will be instrumental to pave the way for the 2nd generation DRUGSFORD compounds and speed-up their development, if needed.

Giving hope to patients suffering from RD
From the start the DRUGSFORD consortium has maintained a close contact to RD patients, especially through the inclusion of Christina Fasser, President of the international patient organisation “Retina International” ( This close contact has provided important input into the project concerning, for instance, the precise and most urgent needs of patients, the design of future clinical trials, patient selection, and the possibility of compassionate use. For the patients suffering from RD, the successful clinical testing of DRUGSFORD compounds would, for the first time, provide a therapeutic option for several disease types with promises for a significantly improved quality-of-life, supporting the active and healthy ageing concept within the framework of the EU programmes. The new company that the DRUGSFORD consortium partners have agreed to set up will greatly facilitate this development. The estimated impact of such drugs and treatments will be important even if the therapy simply delays RD rather than completely stops it, since already this can prolong the patient’s independent and socially constructive life.

Although DRUGSFORD focussed on neurodegenerative diseases of the retina, the potential applications of project results, compounds, and treatments may extend further to neurodegenerative diseases in general. For instance, the compounds and drug delivery technology developed in DRUGSFORD may be used in similar ways to treat or prevent damage to auditory neurons in the inner ear (Jaumann et al., Nat. Med. 18:252-9, 2012) or to spinal cord neurons (Trifunović et al., PeerJ 3:e1189, 2015). Therefore, the resulting nucleotide-based drugs, their prodrug precursors, and their DDS may also yield both novel basic research and therapeutic applications.

List of Websites:
Address of the public website:

Contact details:
Dr. Francois Paquet-Durand
Experimental Ophthalmology
Centre for Ophthalmology
Institute for Ophthalmic Research
Elfriede-Aulhorn-Strasse 5-7
72076 Tuebingen
Phone +49 70 71 / 29 8 74 30