“RESEARCH – CREATE – INNOVATE” INITIATIVE

Project Description

While the overall incidence of cancer mortality has steadily decreased due to improvements in drug delivery technology, the mortality is still increasing in pancreatic cancer (PC). PC has a limited 7% overall surviving rate within 5 years, while almost 30% of the patients die within 2 months from diagnosis. Traditional PC treatments include resection, complemented with radiotherapy and/or chemotherapy. Traditional chemotherapeutics suffers from many constraints, such as severe side effects, nonselective cytotoxicity leading to noncompliance, prolonged treatment, drug resistance, incomplete cure, and low patient quality of life.

Recently, we are trying to shift from traditional to novel drug delivery systems (DDS) by applying nanotechnology to medicine. Although research to identify more efficient drugs is advancing fast, the discovery of novel materials with the required functionality of targeted and efficient drug delivery to PC cells is not progressing at the same rate. PC has the unique property of a high stromal-to-epithelial ratio. Tumor stroma increases the interstitial fluid pressure, thus preventing drugs from penetrating the tissue interstitium. In contrast to other solid tumors where cancer-associated fibroblasts promote tumor growth and angiogenesis, the fibrotic stroma in PC inhibit the formation and the function of blood vasculature, thereby diminishing drug delivery via perfusing blood, leading to poor effectiveness of systemic chemotherapy by using drug nanoparticles as nanocarriers. It is clear that particularly at this tumor it is necessary to apply a local administration technology rather than nanoparticulate DDS. The best localized DDSs are hydrogels. In order to achieve effective and selective local drug delivery through hydrogels, the implanted polymeric carriers forming the hydrogel should have the ability to release the drugs only under cancer tissue conditions, i.e. lower pH and higher temperature, as compared to the conditions of healthy tissues. In addition, the hydrogels should be able to be implanted with the simplest administration way in order to reduce patient morbidity. For these reasons, “smart” polymeric materials are required that present the required self-organization in order to achieve effective encapsulation and selective drug delivery.

Synthetic polypeptides combine the advantages of natural proteins as well as of conventional polymers. They can be prepared in large quantities, are usually biocompatible and most importantly, they present the sophisticated structure and complexity of the natural proteins through the formation of 3D structures such as α-helix, β-sheet. The aim of this project is the synthesis of injectable in situ forming self-healing hydrogels in order to be implanted in a controlled and the simplest way at the vicinity of the cancer tissue and will be pH-stimuli-responsive to become liquid only at the interphase of the cancer tissues and achieve the targeted and directional delivery for the chemotherapeutic drugs. The proof of concept will be shown with preclinical studies. In addition it will be prepared in pilot quantities and will be made a business plan to evaluate the cost of this material to become commercially available.

Recently, the groups that participate in this project have synthesized polypeptidic hybrid polymers that due to their complex macromolecular architecture and the secondary structure of the polypeptidic blocks, form injectable in situ self-healing stimuli-responsive at pH hydrogels. When subcutaneously placed at the vicinity of the PC tissue, they become liquid at the interphase thus releasing the drug selectively towards the cancer tissue, as proved with preclinical in vivo studies. This work has been filed for a Greek Patent. We will optimize this system to respond under the cancer tissue conditions, and deliver selectively the drugs gemcitabine and paclitaxel for the treatment of PC, a highly lethal disease that urgently need an effective DDS.

Object and Objectives of the Project

The aim of the project is the synthesis in pilot plant of novel hydrogels that will transfer in a targeted mode anticancer drugs for the treatment of pancreatic cancer (PC).

PC is a highly lethal disease since the 5-year survival rate is only 7%, while 30% of the patients die after 2 months when diagnosed with this kind of cancer. The traditional treatment of PC includes resection, complemented by radio- or/and chemotherapy. Unfortunately, the current chemotherapeutic methods lead to unwanted side effects such as cytotoxicity to healthy tissues, which lead to long-termed therapies, chemoresistance and low quality of life for the patient. Recently, there is a big effort taking place for the transition from the traditional to novel drug delivery systems by using Nanomedicine. Although the development of novel and more effective drugs for PC treatment is progressing fast, unfortunately, the discovery of novel and effective drug delivery systems is not developed with the same rate.

PC has the unique property of a high stromal-to-epithelial ratio. As a consequence, combined and targeted chemotherapy is required that induces not only apoptosis to tumor cells, but also elimination of stromal fibroblasts. However, in all current clinical trials, the drugs were delivered in an untargeted fashion, inducing severe side effects. The unique PC microenvironment, which diminishes drug delivery to PC cells via perfusing blood, renders treatment with conventional agents more difficult. Due to this exceptional phenomenon, untargeted delivery through nanoparticles will cause drug accumulation in normal cells rather than in PC cells, explaining the higher mortality rate for PC compared to other tumors. It is thus absolutely necessary, especially in PC, to deliver drugs in a targeted fashion, by means of a nanoconstruct with a local administration mode of action such as hydrogel implants.

The best drug delivery systems for PC drug-loaded implants are administered directly at the site of disease, offering the advantages of controlled and prolonged drug release, direct delivery to the site of disease, hence minimizing drug waste and reduced side effects due to the avoidance of systemic circulation of chemotherapeutic drugs. The best approach to achieve local drug release is through polymeric hydrogels because they can achieve effective, selective and sustained release in order to reduce patient morbidity.

Synthetic polypeptides combine the advantages of proteins and of synthetic polymers. They can be produced in large quantities, are biocompatible, biodegradable and they presents the functionality of the natural proteins based on the 3D structures such as α-helix and β-sheet.

The aims of the project are:

1. Synthesis of multifunctional hybrid-polypeptidic hydrogels.
2. Encapsulation of the anticancer drugs gemcitabine and paclitaxel within the hydrogels.
3. The hydrogels will present the property of self-healing and responsive to pH and temperature for the local administration at the vicinity of cancer tissue, with the simplest possible way, through a syringe and the targeted release towards the cancer rather than the healthy tissue.
4. Synthesis of polymers PEO-b-poly(ε-caprolactone) (PEO-b-PCL). Encapsulation of the anticancer drug paclitaxel at the nanoparticles for the encapsulation of the nanoparticles loaded with the drug to the hydrogels.
5. The encapsulation of the nanoparticles loaded with the drug to the hydrogels.
6. Study of the rheological properties of the empty hydrogels as well as of the loaded hydrogels with gemcitabine and nanoparticles with paclitaxel.
7. Toxicological studies of the hydrogels, empty and loaded with the frugs.
8. Production of pilot plant quantities under GMP (Good manufacturing Procedures) conditions to evaluate the cost of a commercial product.

We will optimize the hydrogels for the best possible treatment of PC.

Project Implementation Methodology

Today, most of the materials used as drug delivery systems through hydrogels function only if they are injected within the cancer tissue, in order the lower pH as well as the higher temperature of the cancer tissue to form in situ the hydrogels, which leads to uncontrolled release.

Recently, the groups that participate at this project we synthesized polypeptidic hybrid polymers that due to their complex macromolecular architecture and secondary structure enable them to form injectable and rapidly reconstituted pH-responsive hydrogels. This resulted to achieving a controlled release of chemotherapeutics to the cancerous tissue. This work led to the filing of a Greek Patent with Application Number 20160100179 and International Patent Classification (IPC): A61K47/34, C08G69/36.

In the present proposal, a series of polypeptdic pentablock terpolymers will be synthesized by the NKUA, which were found to form hydrogels and the hydrophilic drug gemcitabine (formulation A) will be encapsulated.

The NKUA will also synthesize a series of hybrid polypeptide pentablock terpolymers where the middle block will be poly(ethylene oxide) instead of poly(lysine) to introduce a temperature dependence in addition to the pH dependence. Here too, a series of polymers with different molecular characteristics will be synthesized and gemcitabine encapsulation will take place (formulation B).

At the AUTH Chemistry Department, nanoparticles encapsulated with the hydrophobic chemotherapy drug paclitaxel (taxol) will be prepared. The nanoparticle-paclitaxel formulation will be integrated into the hydrogels either alone (formulation C) or with an encapsulated drug gemcitabine (formulation D) resulting in a new formulation for the simultaneous administration of two chemotherapeutic drugs that act synergistically. These drugs have already been approved for the treatment of pancreatic cancer and it is expected that the final formulation will have a very high efficacy.

Empty hydrogels and drug-loaded hydrogels (formulation A, B, C and D) will be studied by rheology by ITE to find their strength and shear strength. The hydrogel should liquefy under the shear stress of the syringe plunger, and reconstitute into a hydrogel as soon as it exits the syringe. At the same time, due to the presence of poly(histidine) and poly(ethylene oxide) (mPEG) it will be pH and temperature responsive, for its targeted release in the cancer tissue, with the lower pH and higher temperature, as the lower pH will protonate polyhistidine and it will lose its self-organization due to its secondary structure and the temperature will reduce the solubility and dimensions of mPEG, the self-organization will be lost and the hydrogel will melt. Rheological properties as a function of pH, temperature, amount of water and polymer/drug or polymer/drug/nanoparticle will be studied.

PHARMATHEN will perform the molecular characterization of the hydrogels with size exclusion chromatography, FT-IR and UV-VIS spectroscopy. Also, the release curves of the drugs from the hydrogel will be made as a function of the temperature at 37 °C at pH=7.4, at 40 °C at pH=6.5. A pilot production of the hydrogel under aseptic conditions and good manufacturing practice (GMP) will also be carried out and a business cost plan for its commercial manufacture will be made.

Finally, from the Medical School of Larissa, there will be a toxicological and pharmacokinetic study of selected nanoformulations in animals, activity studies of selected formulations in animal models of cancer and determination of the pharmacokinetic parameters of the selected formulations.

Description of work units and deliverables

A. Synthesis of multifunctional hydrogels and formulations.
 

Pentablock poly(L-lysine)-b-poly(L-histidine-co-γ-benzyl ester of L-glutamic acid)-b-poly(L-lysine)-b-poly(L-histidine-co-γ -benzyl ester of L-glutamic acid)-b-poly(L-lysine), using α,ω-diaminohexane as a difunctional initiator, and the same with a PEO middle block using α,ω-diamino-PEO, as a difunctional initiator and the sequential addition of the corresponding N-carboxy anhydrides.

Formation of gemcitabine hydrogels by dissolving the drug in water and adding it to the solid polymer.

Formation of nanoparticle-entrapped hydrogels with paclitaxel.

Formation of nanoparticle-entrapped hydrogels with paclitaxel and gemcitabine.

Deliverables:

1. Polymers,
2. Protocols for the preparation of polymers and hydrogels with drugs,
3. Hydrogels with encapsulated drugs and nanoparticles, 1 Publication, 4 participations in Conferences

B. Synthesis of PEO-b-PCL copolymers. Encapsulation of paclitaxel to nanoparticles (PEO-b-PCL+PTX).

Synthesis of PEO-b-PCL diblock copolymer via ring-opening polymerization of ε-caprolactone using α-methoxy-o-hydroxy-poly(ethylene glycol). Various parameters will be optimized in order for the final materials to have the desired properties. Paclitaxel will be encapsulated in cross-linked nanoparticles by the oil/water emulsion method and the use of EDTA as a cross-linking agent.

Deliverables:

1. Polymers,
2. Protocols for preparation of polymers and nanoparticles with drugs,
3. Solutions of Nanoparticles with drugs, 1 Publication, 5 participations in conferences.

C. Molecular Characterization of polymers.

The polymers will be characterized physicochemically by NMR spectroscopy, FT-IR, GPC for molecular weight determination, DSC, XRD, swelling study, enzymatic hydrolysis, static and dynamic light scattering. Hydrogels and encapsulated nanoparticles will be studied by SEM, TEM, DLS, FTIR, DSC and XRD.

Deliverables:

1. Molecular characterization of polymers,
2. Molecular characterization of hydrogels and nanoparticles.

D. Rheological behavior of empty hydrogels and hydrogels with entrapped gemcitabine and paclitaxel nanoparticles.

The strength of the hydrogel as a function of the water content, drug content, pH and temperature between the values of cancer and healthy tissues will be studied with Rheology measurements by Prof. Vlassopoulos' group. Shear thinning will be examined to be injectable, self-healing, and formed in situ in the mouse body. The dependence of the self-healing time as well as the power of the hydrogel required will be studied.

Deliverables:

1. Results of rheological studies, mechanical measures, viscosity, self-healing properties.

E. Pharmacological characterization of preparations.

Drug content and drug release rate will be studied and release curves will be made.

Deliverables:

1. Results of pharmacological characterization of hydrogels and nanoparticles.

F. Preclinical in vivo studies of the formulations.

Toxicological and toxicokinetic study of selected nanoformulations in animals, comparative study of toxicity in subacute repeated administration, activity studies of hydrogel formulations in animal models of cancer and determination of the pharmacokinetic parameters of the nanoformulations.

Deliverables:

1. Toxicological profile of selected hydrogel preparations,
2. Efficacy and selection of the most active preparations/ proof of the feasibility of the innovative idea,
3. Pharmacokinetic parameters of the preparations, 5 participations in conferences.

G. Study for the preparation of pilot quantities of the preparations under GMP.

Preparation of hydrogel preparations in quantities of 5 kg under aseptic conditions, and the cost of their preparation will be calculated.

Deliverables:

1. Formulation preparation protocols in quantities of 5 kg
2. Results of the cost of production of pilot quantities and
3. Final report.

Expected results

Cancer is the second leading cause of death worldwide, with no effective treatment currently available, and has rightly been characterized as the scourge of the century. To address this, there is a strong worldwide research interest in the creation of innovative pharmaceutical systems with important clinical applications, such as the detection of cancer in early stages, the targeted release of drugs in cancer cells, and the visualization of the effects of drug action on cancer tissues. This is the aim of the proposed project. To create an innovative formulation by combining recent technological innovations in hydrogels and nanotechnology in order to prepare a novel formulation for local and controlled release of anticancer drugs. The delivery systems designed in the present project with a combination of innovative carriers such as hydrogels and nanoparticles afford more efficient, targeted and controlled release of anticancer drugs.

The importance of the novel drug delivery systems is that they can be injected and remain for a long time at the vicinity of tumors as hydrogels. In this way, the release of anticancer drugs is achieved only towards cancer cells, eliminating the toxic side effects of classical chemotherapy. These synthetic systems are also highly versatile and can be designed to elicit desired drug release kinetics, uptake, and response from the body.

The proposed research and innovation of the PANHYDROMED program covers all the objectives defined by the "RESEARCH - CREATE - INNOVATE" program, and the expected benefits for PHARMATHEN SA and the participating academic institutions are multiple:

• Development and production of novel polymeric carriers such as those proposed which will aim at the targeted and controlled release of active anticancer substances. At the same time, they will optimize the dissolution rate of poorly soluble pharmacological compounds such as paclitaxel.
• Development of know-how to transfer this technique to production level so that commercially available products can be produced.
• Increasing productivity and replacing problematic formulations.
• Improving the position and competitiveness of PHARMATHEN SA in the international market with the production of innovative products.
• Strengthening the link between research and production (obvious with the collaboration between Pharmathen and Universities). Development of synergies and partnerships in fields of common interest.
• Strengthening and specialization of the research potential (doctoral candidates and postgraduate students of the university institutions will be employed to implement the research project).
• Reinforcement of the company's strategic planning at national and European level for expansion, modernization and promotion of existing infrastructure and provided innovative services.

The scientific results of the proposed PANHYDROMED research proposal for the development of anticancer drugs beyond synergy are:

• Multidisciplinary approach. The main researchers act in addition covering a wide range of scientific fields such as Chemistry, Pharmacology, Pharmacy, Medicine, Sales, Marketing etc.
• Improving patients' quality of life (due to the benefits of the drug, patients' quality of life will be greatly improved).
• Maintaining the optimal drug concentration and the increased duration of the therapeutic effect,
• Stabilization of the patient's response to treatment,
• Increase patient compliance,
• Greater safety from the administration of drugs (e.g. avoiding overdose),
• Improving the bioavailability of the drug and the effectiveness of the treatment,
• Reduced costs for the care provider and for the patient himself.