NANOHAT: Development of a safer and more effective sleeping sickness drug.

Lead Research Organisation: King's College London
Department Name: Pharmaceutical Sciences

Abstract

Sleeping sickness or human African Trypanosomiasis (HAT) is a potentially fatal disease caused by the parasite, which is transferred to humans by the bite of an infected tsetse fly. After being bitten, the infected human will experience two stages of disease: the first stage is the blood-based stage of the disease and the second stage affects the central nervous system (CNS), which occurs once the parasite penetrates the brain from the blood. The infected human will die if Stage 2 HAT is left untreated. The blood-brain barrier (BBB) makes the CNS stage difficult to treat because it prevents 99% of all known drugs from entering the brain from the blood, including some anti-HAT drugs. Those anti-HAT drugs that do enter the brain are toxic compounds in their own right and have serious side effects. Pentamidine is a less toxic blood stage drug, which our research has shown has a limited ability to cross the BBB due to its removal by efflux transporters, which are membrane proteins that remove toxic substances from cells. Furthermore transporters are considered essential in the mode of action of pentamidine against the parasites that cause HAT. The objective of this multi-disciplinary study is to use nanotechnology to both improve the delivery of pentamidine to the brain and into the parasite, whilst reducing its side effects.

In doing so, we will create a novel formulation that will encapsulate pentamidine. Specifically, we will use an amphiphilic polymer (a long molecule composed of many repeating units, bearing water-loving and water-hating blocks), which spontaneously self-assembles into small aggregates called micelles. These micelles can be used to encapsulate the drug, thus prolonging its circulation time and protecting it. In addition, these specific polymers (namely, Pluronics) have been shown to interact with efflux transporters and thus allow passage of the drug across the BBB. This indicates that this improved drug formulation has potential to treat Stage 2 disease.

The optimal formulation will be determined using an iterative screening process that will utilise a series of cutting-edge research techniques from various biophysical, pharmaceutical and biological disciplines. The end product of this research will be a formulation which will be ready to be taken to pre-clinical testing for the treatment of Stage 2 HAT.

Technical Summary

Human African trypanosomiasis (HAT or sleeping sickness) is a potentially fatal disease caused by the parasite Trypanosoma brucei sspp. The disease has two stages - a haemolymphatic stage after the bite of an infected tsetse fly, followed by a central nervous system (CNS) stage where the parasite penetrates the brain, causing death if left untreated. The blood-brain barrier (BBB) makes the CNS stage difficult to treat because it prevents 99% of all known compounds from entering the brain, including some anti-HAT drugs. Those that do enter the brain are toxic compounds in their own right and have serious side effects. Pentamidine is a less toxic blood stage drug, which our research has shown has a limited ability to cross the BBB due to its removal by efflux transporters. Furthermore transporters are considered essential in the mode of action of pentamidine against trypanosomes. The objective of this multi-disciplinary study is to use nanotechnology to both improve the delivery of pentamidine to the brain and trypanosomes, whilst reducing its side effects.

Planned Impact

WHO MIGHT BENEFIT FROM THIS RESEARCH?
The proposed studies will generate an optimal Pluronic/pentamidine formulation for treating Stage 2 HAT and should also reduce adverse events associated with pentamidine treatment of Stage 1 HAT. Therefore the primary beneficiaries of this work will be the patients being supported by the National Sleeping Sickness Control Programmes (NSSCPs) and the Non-Government Organisations (NGOs) responsible for HAT control and treatment programmes.

The findings of these studies will also provide valuable information to scientists studying the self-assembly, drug encapsulation and shape and size of resulting aggregates of Pluronics, the efficacy of Pluronics on the ability of pentamidine to cross the BBB, the toxicity and efficacy of the formulations (in vitro & in vivo) and the efficacy of these formulations in live animal models, both nationally and internationally. More generally, the results should be of interest to the greater drug delivery community as Pluronics are currently of significant interest as drug delivery vehicles so all of the findings can be used as a basis for scientists considering using them to deliver different drugs or to treat different diseases.

HOW MIGHT THEY BENEFIT FROM THIS RESEARCH?
Although not a true measure of incidence, analysis of treatment rates for stage 2 HAT published by the WHO (17th Expert Committee on the Selection and Use of Essential Medicines (2009)) indicate that the number of patients diagnosed with and treated for Stage 2 HAT caused by T.b. gambiense is decreasing year on year, but remains steady at ~60% of the annual new infection rate. Thus in 2007 (the latest year for which data are available) 10,473 new cases of T.b. gambiense were detected and 6,461 cases of Stage 2 HAT were treated, yielding a current treatment population of approximately 6,500 per annum, around one fifth of total worldwide cases.

The majority of Stage 2 treatment is moving away from the more toxic melarsoprol, towards either eflornithine, or nifurtimox/eflornithine combination therapy (NECT), which requires 7 days of twice daily intravenous, as well as 10 days of 4 times daily oral therapy. Our solution provides the benefit of being able to treat Stage 2 disease with an existing drug that is currently used to treat Stage 1 disease. It will simplify the treatment regimes and replace eflornithine and NECT therapy in Stage 2 disease. Our combined pentamidine-Pluronic formulation has the potential to provide a single therapeutic entity for the safer, simpler and cost-effective treatment of all Stages of HAT.

More than 98% of all drugs discovered for the brain cannot be used, because they do not cross the BBB. Our solution could be used to re-formulate drugs for CNS delivery or other compartments where access is limited by efflux pumps.

Furthermore, the success of pentamidine against HAT has led to other diamidine compounds being developed. Pentamidine is also used in the treatment of American cutaneous leishmaniasis and pentamidine transporters are expressed in certain Leishmania species, including P-gp-like and MRP-like (pentamidine resistance protein 1) transporters. Pluronics could improve efficacy of other drugs (including diamidine analogues) that target trypanosomes or leishmania.
 
Description Dr Thomas contributed to MRC Translation Research Program Evaluation
Geographic Reach National 
Policy Influence Type Participation in a national consultation
 
Description DPFS-MRC
Amount £10,000 (GBP)
Funding ID MR/K015451/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 05/2014 
End 04/2014
 
Description MRC
Amount £500,000 (GBP)
Organisation Medical Research Council (MRC) 
Department MRC Confidence in Concept Scheme
Sector Charity/Non Profit
Country United Kingdom
Start 09/2015 
End 08/2016
 
Description MRC PhD Studentship
Amount £90,000 (GBP)
Funding ID MRC 2012 DTG - Ref No MR/K500811/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2012 
End 09/2016
 
Description Chan Test 
Organisation ChanTest
Country United States 
Sector Private 
PI Contribution LAY OBJECTIVE: Quantify neurotoxicity potential of drug. The objective of this study is to examine the in vitro effects of Pentamidine Isethionate Salt on cloned hKir2.1 potassium channels (encoded by the human KCNJ2 gene and expressed in HEK293 cells), responsible for IK1, inwardly rectifying potassium current.
Collaborator Contribution Designed study.
Impact We intend to include the data in a toxicity publication.
Start Year 2013
 
Description Cyprotex 
Organisation Cyprotex
Country United Kingdom 
Sector Private 
PI Contribution The data our post-doctoral research assistants acquired at King's College London using human brain endothelial cell and mouse brain endothelial cells and radioactive analyses suggested that a different approach may be needed to test our hypothesis. Hence we linked with Cyprotex (contract research organization) to undertake surrogate blood-brain barrier models (MDCK-MDR) using LCMS analyses. We also used them to analysis biological samples (LCMS) obtained after standard PK studies with our formulations.
Collaborator Contribution Provided prompt delivery of results, and were open to discuss their results and repeat if necessary. We were pleased with the service they provided.
Impact We are intending to publish this research together with the data that we have obtained at King's College London.
Start Year 2013
 
Description Dr Margerita Valero (University of Salamanca) 
Organisation University of Salamanca
Country Spain 
Sector Academic/University 
PI Contribution The Nanohat team designed the study and wrote the application that received the MRC DPFS award.
Collaborator Contribution Dr Valero is our collaborator and provided expertise on the physico-chemical characterization using spectroscopic techniques, as well as advice on pharmaceutical aspects of the formulation development.
Impact We intend to publish the results of this study.
Start Year 2013
 
Description Drugs for neglected disease initiative (DNDi) 
Organisation Drugs for Neglected Diseases initiative (DNDi)
Country Switzerland 
Sector Charity/Non Profit 
PI Contribution Our contact at DNDi is Dr Robert Don who holds overall responsibility for DNDi's discovery research and preclinical development. DNDi and King's College London have signed a confidentiality agreement, that allowed them to advise us throughout the duration of this project. We shared the preliminary application of this project with them.
Collaborator Contribution The mission of DNDi is to develop new drugs, or new formulations of existing drugs, for patients suffering from the most neglected communicable diseases such as trypanosomiasis and leishmaniasis. DNDi and King's College London have signed a confidentiality agreement, that allowed them to advise us throughout the duration of this project.
Impact Advice on IP.
Start Year 2012
 
Description Project Manager 
Organisation Catalyst
Country United Kingdom 
Sector Private 
PI Contribution We contracted a project manager to help manage this project.
Collaborator Contribution Dr Gayle Chapman worked with Dr Sarah Thomas (and the rest of the NANOHAT team) to provide minutes and agendas for the monthly PMGs needed for the NANOHAT MRC DPFS.
Impact Regular reporting to the MRC as per the condition of the MRC DPFS award
Start Year 2013
 
Description in vitro compound profiling custom CNS side effect panel Perkin Elmer 
Organisation Novascreen biosciences
Country United States 
Sector Private 
PI Contribution Designed study. Custom CNS side effect panel
Collaborator Contribution King's College London would like to test 1 compound (pentamidine isethionate salt) in PerkinElmer Discovery Services' (PDS) CNS Side Effect Panel (CNS SEP)
Impact We propose to publish this data in an open access publication.
Start Year 2013
 
Description An invited oral presentation Trasporters AAPS-Baltimore 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact DISSEMINATION/ESTEEM Invited to present research (speaker) at AAPS transporters meeting -April 2016. Baltimore, USA.
Year(s) Of Engagement Activity 2016
URL https://www.aaps.org/Transporters/
 
Description An invited oral presentation-Parasites meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact DISSEMINATION/ESTEEM Invited to present research (speaker) at Parasites Infection Meeting-June 2016 London 2016.
Year(s) Of Engagement Activity 2016
 
Description Publication of the research in a pre-publication journal prior to submission of the mansucript to a peer-reviewed journal 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Preprints are an important step toward a more open and transparent peer review process -- one that brings with it tremendous benefits for both individual authors and the broader scientific community.
To disseminate the results and outcome of the award as rapidly as possible.
Year(s) Of Engagement Activity 2019
URL http://biorxiv.org/cgi/content/short/573329v1
 
Description The CAAT in the HAT and the BBB 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact Approximately 20 under-graduate/post-graduate students from King's College London attended this event and it stimulated interest in a research career path.

Students wished to undertake projects in my laboratory.
Year(s) Of Engagement Activity 2012
 
Description Treating Trypanosomal Infection of the CNS with Small Molecule Therapy. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Raised profile of my research activity and the NANOHAT MRC DPFS project.

I was asked to review a grant.
Year(s) Of Engagement Activity 2014
URL http://www.ohsu.edu/xd/research/centers-institutes/neurology/blood-brain-barrier/news-and-events/mee...
 
Description Women in Science 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact To demonstrate my awareness of my career development and strategy for promotion to reader as a woman with caring responsibilities.

Promoted discussion regarding possible career paths.
Year(s) Of Engagement Activity 2014