Validating alternative models to cats and dogs for heartworm drug testing
Lead Research Organisation:
Liverpool School of Tropical Medicine
Department Name: Tropical Disease Biology
Abstract
Heartworm is a parasitic worm disease in cats and dogs in many areas of the world. More infections are being identified in Northern Europe and cases have been identified in the UK. Left untreated, heartworm is potentially lethal. Current drugs used to prevent heartworm from infecting the heart are not working as well because of the development of drug resistant parasites. Drugs used to treat heartworms cause side effects which can be lethal. New heartworm drugs are being developed by animal healthcare companies. Currently, we don't have any alternative way of testing new drugs other than to experiment on cats or dogs. Sometimes cats or dogs will become very ill as a result of being infected with heartworm for the purposes of testing drugs.
In this project we will develop cell culture and mouse alternatives to cats and dogs to support the early larval growth of heartworm. We will select the best cultures and mouse strain and check to see whether two different types of drug can give the expected effects in these new models.
When we get to this point in the project we will advocate the adoption of these alternatives to a range of important potential end-users including testing laboratories and animal health care companies.
We calculate that these alternative models will reduce overall numbers of animals used in heartworm drug testing. We also estimate that using mice instead of cats or dogs in drug testing will refine testing by avoiding long duration infections that can induce severe pain and suffering.
In this project we will develop cell culture and mouse alternatives to cats and dogs to support the early larval growth of heartworm. We will select the best cultures and mouse strain and check to see whether two different types of drug can give the expected effects in these new models.
When we get to this point in the project we will advocate the adoption of these alternatives to a range of important potential end-users including testing laboratories and animal health care companies.
We calculate that these alternative models will reduce overall numbers of animals used in heartworm drug testing. We also estimate that using mice instead of cats or dogs in drug testing will refine testing by avoiding long duration infections that can induce severe pain and suffering.
Technical Summary
Heartworm, caused by the filarial parasites Dirofilaria immitis and D. repens, is a disease of veterinary importance in cats and dogs. Left untreated it can cause life-threatening morbidities including congestive heart failure and cardiopulmonary embolism. The annual heart worm market is estimated to be in excess of US$150 million. New heartworm anti-infectives are being developed by animal health companies due to the emergence of resistance to current prophylactics and poor treatment outcomes with available adulticides. Currently there is no alternative to testing novel compounds in experimentally infected cats or dogs.
In this project we will develop in vitro and in vivo alternatives to cats and dogs to propogate D. immitis larval development. We will subsequently validate their utility as heartworm anti-infective screens using reference larvicides (ivermectin) or growth inhibitors targeting the endosymbiont, Wolbachia (doxycycline). Our approach exploits recent advances in mammalian cell co-culture systems supporting filarial larval development and the establishment of new models of human filariasis in compound immunodeficient mice. Proof-of-concept data has been generated that D. immitis infectious larvae can be generated by membrane feeding Aedes aegypti mosquitoes in our laboratory with D. immitis microfilariae shipped from veterinary sources in the USA. Further we have been able to isolate viable L4 larvae from subcutaneous tissues after inoculation of infectious larvae in a severe combined immunodeficient gamma chain knockout mouse strain.
Validation and future adoption of these alternative models will reduce overall numbers of animals used in heartworm translational research. Adoption of a mouse preclinical model will also refine animal use by substituting the majority of procedures causing chronic infection, pathology, severe pain and distress in cats or dogs with less severe, short term infections in mice which avoid pathology.
In this project we will develop in vitro and in vivo alternatives to cats and dogs to propogate D. immitis larval development. We will subsequently validate their utility as heartworm anti-infective screens using reference larvicides (ivermectin) or growth inhibitors targeting the endosymbiont, Wolbachia (doxycycline). Our approach exploits recent advances in mammalian cell co-culture systems supporting filarial larval development and the establishment of new models of human filariasis in compound immunodeficient mice. Proof-of-concept data has been generated that D. immitis infectious larvae can be generated by membrane feeding Aedes aegypti mosquitoes in our laboratory with D. immitis microfilariae shipped from veterinary sources in the USA. Further we have been able to isolate viable L4 larvae from subcutaneous tissues after inoculation of infectious larvae in a severe combined immunodeficient gamma chain knockout mouse strain.
Validation and future adoption of these alternative models will reduce overall numbers of animals used in heartworm translational research. Adoption of a mouse preclinical model will also refine animal use by substituting the majority of procedures causing chronic infection, pathology, severe pain and distress in cats or dogs with less severe, short term infections in mice which avoid pathology.
Planned Impact
New heartworm drugs are needed due to drug-resistance and poor treatment outcomes with current medications. Presently, there are no alternatives to experimental cat or dog infections to test drugs.
By analysing published studies that provided procedural data in heartworm experiments between 2015-2017, we identified 163 lab-reared cats and 431 dogs were used (a total use of 594). Of the animals used, 59% (325) were used in drug testing. This may be an underestimate of total use due to lack of data sets in the public domain. We estimated that infection studies give rise to severe adverse events in cats or dogs (such as acute cardiopulmonary embolism) in ~20% of cases due to some animals suffering the effects of unpredictable, high worm burdens following experimental infections. This equates to an estimated 40 procedures per annum causing severe harm.
This project addresses a solution to both reduce and refine current experimental use of cat and dogs in heartworm anti-infective testing at the preclinical level. At the completion of the project we aim to have developed an 'assay-ready' screening cascade encompassing an in vitro screen and a murine small animal alternative research model to evaluate efficacy of new heartworm preventive drugs.
The establishment of an in vitro screen and in vivo testing in a short-term heartworm murine model will reduce overall animal use, reduce costs and improve the quality of testing data. Some dog or cat experiments are still required but numbers will be reduced. Mice will be used as an initial in vivo screen which reduces overall procedures. This is because:
1. an in vitro screening system and pre-existing murine pharmacokinetics (PK) can be utilised in industry-standard in silico modelling techniques to interrogate likely in vivo efficacy. Only candidates passing these criteria will be advanced into animal testing, thereby preventing animal testing for a proportion of candidate drugs.
2. Use of murine PK-Pharmacodynamic modelling will allow for a more accurate calibrated dose selection to test efficacy, thereby reducing the necessity for multiple dose groups.
3. the mouse model will reduce intra-group variation in primary endpoint analysis (accurate worm yield) and in PK exposures within an immunodeficient mouse model, compared with large outbred animals. This will reduce group size necessary to provide appropriately powered efficacy data.
4. The requirement of repeat experiments due to equivocal data should be avoided or reduced.
The project will also refine animal use for heartworm testing by replacing many procedures likely to cause severe harm in cats or dogs with shorter term mouse testing avoiding severe harm.
We hypothesise that implementing a new screening cascade may impact on total animal use by 54% (176 over a three-year period). We derive these reductions by considering the impact of a rigourous in vitro screen and an in silico prediction of efficacy utilising pre-existing murine PK. This could de-prioritise an estimated 7/10 drug candidates before the point of in vivo testing, avoiding both dog / cat PK experiments and efficacy studies. Further, by substituting cats/dogs with mice in an initial in vivo screen, we will reduce overall animal use by an additional 25%. By reducing cat/dog experiments and utilising a more short-term mouse model, we would avoid causing severe harm by as much as 59% (192 cats/dogs over three years). We predict for every 10 drug candidates assessed in our alternative models, only 2 will meet all performance criteria to enter cat/dog experiments.
We will utilise our extensive networks with pharmaceutical industrial end users and a major heartworm testing laboratory to adopt the new alternative models to test novel heartworm drug candidates. We will lobby for change in practise in other major testing labs by arranging collaborative and scientific exchange visits and by attending focused European and American Heartworm Society symposia.
By analysing published studies that provided procedural data in heartworm experiments between 2015-2017, we identified 163 lab-reared cats and 431 dogs were used (a total use of 594). Of the animals used, 59% (325) were used in drug testing. This may be an underestimate of total use due to lack of data sets in the public domain. We estimated that infection studies give rise to severe adverse events in cats or dogs (such as acute cardiopulmonary embolism) in ~20% of cases due to some animals suffering the effects of unpredictable, high worm burdens following experimental infections. This equates to an estimated 40 procedures per annum causing severe harm.
This project addresses a solution to both reduce and refine current experimental use of cat and dogs in heartworm anti-infective testing at the preclinical level. At the completion of the project we aim to have developed an 'assay-ready' screening cascade encompassing an in vitro screen and a murine small animal alternative research model to evaluate efficacy of new heartworm preventive drugs.
The establishment of an in vitro screen and in vivo testing in a short-term heartworm murine model will reduce overall animal use, reduce costs and improve the quality of testing data. Some dog or cat experiments are still required but numbers will be reduced. Mice will be used as an initial in vivo screen which reduces overall procedures. This is because:
1. an in vitro screening system and pre-existing murine pharmacokinetics (PK) can be utilised in industry-standard in silico modelling techniques to interrogate likely in vivo efficacy. Only candidates passing these criteria will be advanced into animal testing, thereby preventing animal testing for a proportion of candidate drugs.
2. Use of murine PK-Pharmacodynamic modelling will allow for a more accurate calibrated dose selection to test efficacy, thereby reducing the necessity for multiple dose groups.
3. the mouse model will reduce intra-group variation in primary endpoint analysis (accurate worm yield) and in PK exposures within an immunodeficient mouse model, compared with large outbred animals. This will reduce group size necessary to provide appropriately powered efficacy data.
4. The requirement of repeat experiments due to equivocal data should be avoided or reduced.
The project will also refine animal use for heartworm testing by replacing many procedures likely to cause severe harm in cats or dogs with shorter term mouse testing avoiding severe harm.
We hypothesise that implementing a new screening cascade may impact on total animal use by 54% (176 over a three-year period). We derive these reductions by considering the impact of a rigourous in vitro screen and an in silico prediction of efficacy utilising pre-existing murine PK. This could de-prioritise an estimated 7/10 drug candidates before the point of in vivo testing, avoiding both dog / cat PK experiments and efficacy studies. Further, by substituting cats/dogs with mice in an initial in vivo screen, we will reduce overall animal use by an additional 25%. By reducing cat/dog experiments and utilising a more short-term mouse model, we would avoid causing severe harm by as much as 59% (192 cats/dogs over three years). We predict for every 10 drug candidates assessed in our alternative models, only 2 will meet all performance criteria to enter cat/dog experiments.
We will utilise our extensive networks with pharmaceutical industrial end users and a major heartworm testing laboratory to adopt the new alternative models to test novel heartworm drug candidates. We will lobby for change in practise in other major testing labs by arranging collaborative and scientific exchange visits and by attending focused European and American Heartworm Society symposia.
People |
ORCID iD |
| Joseph Turner (Principal Investigator) | |
| Mark Taylor (Co-Investigator) |