Main Findings

Work Package 1: Nanocarrier design, development and characterization

 

Objectives

  • Nanocarrier selection and development nanotechnologies (Phases A-F)
  • Nanocarrier characterization (Phases A-F)
  • Nanocarrier optimization (Phases B-D)
  • Final processing of nanocarriers and pharmaceutical presentation (Phases D-E)
  • Nanocarrier production for advanced clinical and GLP toxicology studies

Work plan and conclusions

The activities of this work package resulted in the development of hundreds of different nanocompositions. Out of these, a total of 13 nanocarrier prototype families fulfilled the TRANS-INT selection criteria and progressed to WP2 for peptide loading. These prototypes are based on well-characterised polymers (e.g. chitosan, arginine-rich polypeptides amongst others), lipids, oils, and in some cases, inorganic materials, which are organized according to different nanostructures (Figure 1). The preparation methods were selected according to the criteria of simplicity, mildness and scalability and were adapted to the specific composition of each prototype. These methods include ionic gelation, complexation, self-assembly and solvent displacement. These methodologies also allowed the incorporation of different fluorescent markers into the nanocompositions in order to enable their mechanistic evaluation and in vivo tracking in subsequent work packages. In this regard, maintaining the original physico-chemical properties of the carrier upon labelling, and avoiding the leakage of the fluorescent dyes over time, were identified as important challenges.

 

Fig 1

Fig. 1. Schematic representation of the different nanostructures

 

This WP also involved the determination of the mucoadhesion and mucodiffusion properties of the TRANS-INT prototype families using a series of different analytical techniques: dynamic light scattering (DLS), microfluidics, capillary-based methods, multiple particle tracking (MPT) and fluorescence recovery after photobleaching (FRAP). As an example, the set-up of the microcapillary technique is illustrated in Figure 2.

Overall, the conclusions that could be extracted from the different techniques are:

- The surface properties of the nanocarriers and, notably the presence of polyoxyethylene coatings, reduce the interaction with the mucus and have a positive effect on the mucodiffusion of the nanocarriers.

- The size of the nanocarriers has a significant influence on their mucodiffusion properties, with smaller size nanocarriers (less than 100 nm) being more mucodiffusive than the larger size nanocarriers.

- The positive charge of the nanocarriers has a negative impact on their mucodiffusion behaviour.

All the mucodiffusion techniques analysed in the consortium have specific “pros” and “cons” (mostly simplicity vs. specificity) and the interpretation of the data obtained in mucin or mucus should be undertaken cautiously. In our opinion, the results of these studies should be analysed jointly with those obtained in the in vitro cell culture studies and also in the in vivo studies. Only the integrated analysis of the in vitro-in vivo behaviour could provide insights on the importance of the mucodiffusion properties on the in vivo performance of the nanocarriers.

 

Fig 2

 

Fig. 2. Experimental set-up of the microcapillary technique used for measuring the mucodiffusion of fluorescent nanocarriers

 

Work Package 2: Formulation of peptide drug candidates

 

Objectives

  • Peptide characterization and stability profiles
  • Development of peptide-loaded nanoparticle formulations
  • Evaluation of the peptide release and in vitro/ex vivo stability of the peptide-loaded nanocarriers
  • Optimization of the peptide-loaded nanocarriers
  • Stability of peptide-loaded nanocarriers during storage
  • Final processing of nanocarriers, pharmaceutical presentation, and stability

 

Work plan and conclusions

Work package 2 (WP2) had a central role in the consortium as it linked the carriers selected in WP1 to the testing of peptide-loaded prototypes in the downstream work packages.

Candidate Peptides. The peptides available to the consortium were focused around diabetes with three types of insulin, and two GLP1 analogues, but also included a pain-relieving peptide, labyrinthopeptin. This range of peptides was introduced and the range of physical chemical properties of peptides expanded as the understanding of the formulation challenges increased. Each of the peptides were characterised and suitable assays were developed by and shared with partners in the form of SOPs.

The selection of formulations for peptide loading were based on initial nanocarrier prototype families (PFs) introduced and evaluated in WP1. These included a range of different types of nanocarriers such as nanocapsules (PF 1, 2, 3 from USC and PF4 from UA), nanoparticles (PF5-USC and PF11-UCLouvain), nanocomplexes (PF6-USC, PF7-UCD) or micelle-like systems (PF8-UCLondon, PF9-UCC); two additional prototypes were introduced later on in the project by USC and UCD. 

An early learning outcome for the consortium was the need to change the emphasis of the initial nanocarrier characterisation from WP1 to WP2. This was necessary as many of the nanocarriers showed a peptide-dependent change of properties from empty to peptide loaded carriers.

To ensure the selection of the most promising prototypes for downstream testing, selection criteria and assays were developed and shared as standard operating procedures (SOP) to allow ranking within and between PFs.  The selection criteria were based on the physiology of the GI tract, on the oral peptide delivery literature as well as on the pharmaceutical and commercial requirements. These criteria included minimal peptide loading efficiency and final loading values, in addition to adequate stability and release profiles in simulated intestinal media, in the presence of enzymes. Specific assays, e.g. level of peptide encapsulation, also needed to be adapted according to the individual carrier systems.

Overall, prototype development was prioritised based on the hypothesis that good candidates should elicit the following properties: (i) small particle size (less than 300 nm, preferably less than 100 nm) (ii) negative/neutral surface charge (iii) high peptide loading efficiency (>50%, targeted value 100%) and final loading (>1%, targeted value 20%) (iv) protection and controlled release of the associated peptide upon exposure to simulated intestinal fluids (v) adequate mucus penetration. Furthermore such carriers should not be altered upon their incorporation in the final dosage forms (freeze-dried powders and SMPills) in terms of the above-indicated parameters.

By combining the various PFs and peptides the consortium developed and characterised hundreds of formulations, which were ranked by applying these selection criteria within each PF to allow the selection of the most suitable composition and processing conditions.

Interestingly, the characterisation strategy also highlighted challenges such as the issue of encapsulation of very water soluble peptides in the various lipid based carrier systems. In addressing this, the consortium also introduced a number of new PFs using alternative encapsulation strategies at a later stage of the development. Ultimately around 13 prototypes were identified for initial in vivo evaluation with a number of different carriers showing promise in various models.

The consortium proceeded with further development based on the assumption that the most convenient dosage form would involve a dry formulation that would be used in a tablet or capsule. Furthermore, for most peptides some protection in the stomach will be beneficial even though nanocarriers can provide some degree of protection.

In addition to the scale up, the preparation of final dosage forms therefore included the preparation and characterisation of a dry peptide loaded nanoparticle powder. In addition, as a potential gastro-protective dosage form, the consortium evaluated up to 5 prototypes formulated according within the SmPill® technology. This technology was proven to be capable of processing liquid suspensions as well as dried formulations. The main limitation was associated with the degree of dilution that was necessary for the nanocarriers to be adequately processed. As a consequence, only nanocarriers with a final loading higher than 20% could be used for this type of formulation. Alternatively, the peptide-loaded nanocarriers could be freeze-dried and incorporated into cellulose-coated enteric gelatine capsules. 

 

Work Package 3: In vitro mechanistic and toxicity studies

 

Objectives

  • Evaluation of cellular toxicity and functionality
  • Evaluation of the interaction of nanocarriers with the intestinal epithelium and transport of the associated peptides

Work plan 

When encapsulating a drug/peptide into a nanosized drug delivery system, the nature of the nanoparticle composition, the geometry and the surface properties can altogether affect interactions with cells. Therefore, the toxicity screening of nanocarriers designed and formulated in WP1 and WP2 were evaluated in vitro in an intestinal cell line, Caco-2, using validated assays: lactate dehydrogenase (LDH) release, ATP content, neutral red assay, MTS cell proliferation assay. All of the prototype families were tested.  The cytotoxicity studies indicate that most of the prototypes tested induce a very low to low cytotoxicity with EC50 values ranging from 0.1 to >8 mg/ml. A toxicological screening of nanocarriers was also carried out based on cellular immunological responses. The prototypes tested did not affect dendritic cells at non-cytotoxic concentrations.

The understanding of the mechanisms involved in nanoparticle interaction and transport across the intestinal barrier and the factors limiting these processes was critical in order to improve the design of nanocarriers and their performance. Flow cytometry and fluorescence microscopy were employed to quantify and visualise the uptake of the nanoparticles by the intestinal cells, using the Caco-2 model cell line. The results showed that the nanoparticles’ composition strongly influenced the interactions with intestinal cells and the mechanisms involved in nanoparticle cell internalisation. The transport of nanocarriers across Caco-2 monolayers was evaluated by assessing either the fluorescence associated with the nanocarriers detected or the peptide quantified by the LCMS/MS technique. The apparent permeability coefficient measured in terms of fluorescent molecule and peptide transport was generally low. However, the accumulation of the nanocapsules and associated protein in the monolayer was significant and variable depending on the prototypes. For example, insulin associated to arginine-rich nanocarriers was highly internalized (up to 80%), however the transport across the monolayer was always lower than 4%. On the other hand, the capacity of the nanocarriers to modify the trans-epithelial electrical resistance (TEER) was variable and dependent on the nanocarrier. Positively charged prototypes, i.e. chitosan or protamine nanocarriers, led to a transient decrease of the TEER, whereas other neutral carriers did not cause a significant modification of TEER.

In a second step, the analysis of a reduced number of prototypes was performed in the ex-vivo human intestinal model (Ussing chamber). Visualization of nanoparticle interactions with tissue was achieved using laser scanning confocal microscopy (LSM), epifluorescence microscopy, and structured illumination super resolution microscopy (SIM). The results showed different levels of interaction of the prototypes with the intestinal mucosa, which could be classified as (i) no permeation, (ii) weak permeation and (iii) active endocytosis. The first category included Polyarginine nanocapsules, EudragitÒ nanocomplexes, and Nanostructured Lipid Carriers (NLC) that showed pronounced mucus binding. The second category consisted of prototype family 3 (Protamine nanocapsules), which showed mucus binding but could also be detected in the tissue and exhibited some uptake. The third category comprised PGA-PEG/r8-glulisine nanocomplexes, which were mucodiffusive (Figure 3). These nanoparticles were highly endocytosed by the enterocytes. 

 

 Fig 3

 

Fig. 3. The nanoparticles were endocytosed by the enterocytes of the intestinal epithelium and showed a small but measurable permeability. (Nanoparticles in green, nuclei in blue)

 

The comparative analysis of these data with those obtained in the Caco-2 model cell line indicates some discrepancies. This could be attributed to the absence or limited presence of mucus in the monolayers. The only two prototypes that behaved comparatively were PF 6 (PGA-PEG/r8-glulisine nanocomplexes), which showed a high internalization in both models, as well as PF 12 (undisclosed for IP reasons). This could also be related to the good mucodiffusive properties of these prototypes.

Conclusions

The capacity of nanocarriers to facilitate the transport of the associated peptide across the epithelium was, in general, limited. This could be attributed to the fact that the nanomaterial and the associated peptide remain associated to the epithelium for a certain period of time. This accumulation of the nanocarriers and associated peptides within the intestinal mucosa might be of potential interest for the treatment of GI disease.

 

Work Package 4: Preliminary in vivo mechanistic and toxicity studies

 

Objectives

  • Evaluation of particle interaction with epithelial barriers and biodistribution
  • Preliminary immunological evaluation

Work plan 

UCLondon carried out studies with fluorescently-labelled nanocarrier Prototype 9 (GCPQ micelles) showing convincing qualitative evidence of interaction and uptake by the mouse small intestine following gavage (Figure 4). This prototype affected expression of the tight junction protein, ZO-1, in Caco-2 cells and in isolated mouse gut loops, suggesting interaction with the paracellular pathway. 

 

Fig 4

 

Fig. 4. Ex vivo confocal laser scanning microscopy in mice instilled with Prototype 9 labelled with Texas Red showing adherence to the mucus layer and permeating between the villi (left), around the intestinal brush border, taken up by the goblet cells, within the lamina propria and in the endothelium (Right). Blue: DAPI; Red: GCPQ-Texas Red particles; green: Tomato lectin. BB: brush border, GC: goblet cell, EC: epithelial cell, BM: basolateral membrane, LP: lamina propria, EnC: endothelial cell.

 

ECAMRICERT and IOV attempted similar studies in rats.Overall, the fluorescence imaging studies showed a variable interaction of the different prototypes with the intestinal mucosa. Polyarginine nanocapsules showed a measureable interaction with the epithelium, whereas such interaction could not be determined for protamine nanocapsules. On the other hand, the NLC prototype was found to interact and remained widely distributed along the intestine for extended periods of time.

UCD used jejunal and colonic instillations in rats under anaesthesia to measure the localisation of particles, as well as the PK/PD effects of seven prototype peptide-loaded carriers. Five fluorescently-labelled prototypes were located to different degrees in intestinal mucus, adhering to the epithelium, and along the sides of microvilli, however several prototypes were detected in rat jejunal epithelia. In the PK-PD studies, two prototypes gave positive data.  Prototype 12 reduced blood glucose and delivered peptides in 6/6 rats (Figure 5), and in a separate batch, in 3/4 rats. This prototype has process reproducibility issues and is being further optimised.  Prototype 8 similarly induced a PD response, accompanied by measurement of peptide in plasma, and a relative bioavailability of 5.5%.

In vivo biodistribution studies were also performed at the USC using radiolabelled nanocarriers. As an example, Technetium-99m (99mTc) labelling was applied to Prototype 6: PGA-PEG/R8-glulisine nanocomplexes (USC). The SPECT-TC imaging biodistribution study concluded that the 99mTC-labelled PGA-PEG/R8-glulisine were retained in the small and large intestines for up to 26 hrs, in contrast to the control that remained mainly in the stomach (Figure 6). However, no significant radioactivity could be observed in the rest of the body, a fact that suggests that the particle interacts with the intestinal mucosa and remains at that level, without systemic absorption, even after extended periods of time.

 

Fig 5

 

Fig. 5. PD profile of rats dosed with Prototype 12. Doses were 1 IU/kg (s.c.), 50 IU/kg (instillations)

 

Fig 6

 

Fig. 6. In vivo tissue distribution of the orally administered 99mTc- PGA-PEG/R8-glulisine (ENCPs)

 

IOV and ECAMRICERT investigated the local mucosal immune response of mice treated with several peptides / prototypes dosed acutely and also once-a-day for 28 days.  No changes in gross epithelial histology were seen, although there were some increases in lymphocytes. These data are important as intestinal immune response studies have not been examined before and the negative data underscores the benefit of using excipient and polymer materials with a history of use in man. 

Conclusion

Overall, the nanocarriers have a deeper or more superficial interaction with the intestinal mucosa depending on their composition. In some cases, the interaction was important enough to prolong the permanence of the nanocarriers in the mucosa for long periods of time and generate PK/PD responses. However, no translocation of the particles across the intestine of rodents could be observed. Despite their internalization, specific nanocarriers showed an adequate biocompatibility.

 

Work Package 5: In vivo pharmacokinetic/pharmacological evaluation of nanomedicines

 

Objectives

  • In vivo evaluation of the pharmacological profile
  • In vivo evaluation of the drug pharmacokinetic profile

 

Work plan

Insulin formulations representing different prototypes and technologies from various institutions were tested at USC, UCD and Sanofi for evaluation of in vivo efficacy and PK/PD relationship. The formulations were tested in rat models of direct intestinal instillation in normoglycemic and diabetic rats under both, anaesthetised and conscious conditions. The effect on blood glucose was measured and compared to that of subcutaneously injected insulin. Whenever possible the PK of intestinally administered insulin was also determined by a specific ELISA assay, for human insulin, or by a specific LC-MS/MS method, for insulin glulisine.

In general we found that several insulin-loaded nanoformulations elicited a statistically significant glucose response following either intra-duodenal or intra-jejunal administration. Although this response was modest (in most instances in the range of a 20-30 % decrease when tested in non-diabetic animals), it is in agreement with the best data observed in the literature in normoglycemic rats.

Some of the nanoformulations tested did not lead to a significant glucose-lowering response following either intra-duodenal administration. These results were in contradiction with what was expected from the in vitro experiments performed in the Caco-2 model cell line and in human intestinal tissue; in which a very important penetration of the nanoparticles and also of the associated insulin was observed.

When tested in STZ-diabetic animals it appears that the tested nanoformulations exhibited a greater decreasing effect on glucose levels as compared to that shown in normoglycemic animals. For example, the results presented in Figure 7 (Prototype 13, insulin nanocomplexes) show the capacity of the administered formulation to exhibit an adequate response.

Despite the positive in vitro data (stability, control release, caco-2 permeability, etc.) obtained for the tested prototypes, it may be that these in vitro parameters have a limited predictive value of the performance of oral peptide formulations.

Conclusion

These data indicate that the efficacy of the formulations was different in different animal models. The non-anaesthetized rat diabetic model (USC) provided the most significant responses, whereas the anaesthetized rat model (Sanofi R&D) did not show any specific response nor any concentration of insulin in blood up to 4 hrs after inoculation. Whether one experimental protocol is more meaningful than the other remains to be elucidated.

Irrespective of the influence of the animal model on the overall output of this WP, it should be noted that these results are somehow in contradiction with what was expected from the in vitro experiments performed in the Caco-2 model cell line and in human intestinal tissue and the in vivo biodistribution studies. In these models, for some specific formulations that did not give a significant response, a very important penetration of the nanoparticles and of the associated insulin was observed.

Two main hypotheses have been formulated to explain these data so far: (i) The positive in vitro data (stability, controlled release, caco-2 permeability…) obtained for some prototypes may have a limited predictive value of the performance of oral peptide formulations in vivo; (ii) The high variability in the response to insulin and the differences between the animal models may be responsible for the highly variable responses observed for all formulations. In this sense, it is important to keep in mind that in order to draw a clear conclusion, it will be necessary to perform the experiments in large animal models. This evaluation will now be performed for selected prototypes after the end of the TRANS-INT project.

 

Fig 7

 

Fig. 7. Blood Glucose levels represented in % in relation with basal levels after intraduodenal administration of Prototype 13 to STZ-induced diabetic rats

 

Work Package 6: Preclinical efficacy, toxicological, immunological evaluation

 

Objectives

  • In vivo advanced preclinical pharmacokinetic evaluation
  • In vivo advanced immunological evaluation
  • In vivo exploratory and GLP-toxicity evaluation

 

Work plan and conclusions

This WP provided preliminary preclinical data regarding the efficacy of one selected prototype in the pig model. Experiments to fulfil the task associated with this WP are still under way and no conclusive data can be reported at this stage. Nevertheless, these data are expected to be published in the form of scientific publications.

 

Work Package 7: Training and education

 

Objectives

  • Development of a modular education program in oral nanomedicine
  • Organisation of a young innovative debating school to discuss advances in oral nano-drug delivery
  • Development of a training program involving personnel exchange between the member institutions

 

Work plan and conclusions

Five training events at annual meetings, and formal education in nanomedicines linked to EU training activities in other consortia, namely NANOFAR and COST actions, were provided.

At the annual meetings thematic teaching was on the following topics: Lipid-based systems, peptide synthesis and mass spectrometry; quality and translation of nanocarriers, in vivo imaging; gut microbes; regulation of biotech medicines. In 2017, a pre-meeting workshop was organized in association with the Spanish-Portuguese Local Chapter of the Controlled Release Society Inc. (CRS).

Continuing professional development: workshops on intellectual property, ethics in research, presentation skills, and on job interviews were provided by partners with the appropriate expertise.

Students and postdocs debated the merits of their data in poster and oral sessions at the annual meeting and also at the “Crossing Biological Barriers” conference in Dresden, 9-11th November 2015.

Journal clubs were run at the annual meeting and debated: “Delivery of peptides to the blood and brain after oral uptake of quaternary ammonium palmitoyl glycol chitosan nanoparticles” Lalatsa A, et al. 2012;9:1764-74; “Transepithelial transport of Fc-targeted nanoparticles by the neonatal fc receptor for oral delivery”. Pridgen EM, et al Sci Transl Med. 2013 5(213):213ra167; “Overcoming the diffusion barrier of mucus and absorption barrier of epithelium by self-assembled nanoparticles for oral delivery of insulin”. Shan W, et al. ACS Nano. 2015 9(3):2345-56; “Interaction with mixed micelles in the intestine attenuates the permeation enhancing potential of alkyl-maltosides”. Gradauer K. et al. Mol Pharm. 2015;12:2245-53.

The student and staff exchange programme was very successful and, over the five years, most partners sent and received staff. There were 25 lab visits.

Numbers of PhDs associated with TRANS-INT:  18, of which 10 were fully funded by TRANS-INT and have had their PhDs awarded/pending. 25 postdocs passed through the programme.

The education programme was very successful and achieved all its deliverables.

 

Work Package 8: Exploitation and Dissemination

 

Objectives

  • To define and implement an integrated strategy for TRANS-INT dissemination and exploitation
  • To regularly inform all stakeholders about TRANS-INT and the project results
  • To promote the (use of) TRANS-INT results and advertise the benefits of the project

 

Work plan and conclusions

An Exploitation and Dissemination plan was prepared and implemented for the TRANS-INT project. The plan described how TRANS-INT would make optimal use of the tools available for dissemination and how TRANS-INT intended to screen all of the results to ensure optimal exploitation.

The project website was created and regularly updated in order to keep the stakeholders informed about TRANS-INT’s activities and the results achieved.

The benefits and results of the TRANS-INT project were promoted through:

  • Implementation of e-tools, such as e-brochures, e-newsletters and e-posters;
  • Participation in the joint “Crossing Biological Barriers” conference with the ALEXANDER (FP7) and COMPACT (IMI) projects; TRANS-INT partners participated actively with 7 oral presentations, 2 posters and with the chairing of 4 sessions;
  • Publication of a themed issue in November 2016 entitled Oral delivery of peptides: opportunities and issues for translation (The EU FP7 TRANS-INT Consortium) in the Advanced Drug Delivery Reviews journal. The issue was edited by Prof. Alonso and Prof. Brayden and contained 12 review articles.

A considerable number of dissemination activities took place at national and international level, including 28 articles, 6 book chapters, 35 conference abstracts and 9 PhD theses.

Exploitation of results was successfully achieved with three patent applications submitted so far and a fourth application in preparation.

As the consortium’s scientific activities are now completed, the dissemination will continue in 2017 to ensure maximum visibility of the project and full exploitation of the results achieved.


 

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