- Email: [email protected]
Current strategies in the search for novel antiparasitic agents
ELSEVIER
International Journal for Parasitology 29 ( 1999) 95- IO3
Current strategies in the search for novel antiparasitic
agents
Michael J. Witty* Animal
Health
Discot>ery,
Pfizer
Central
Research,
Ramsgate
Road, Sandwich,
Kent CT13 9NJ.
UK
Received 24 March 1998; accepted 7 October 1998
Abstract
The market for antiparasitic products comprises the largest segment for sales of livestock and companion-animal healthcare agents. Despite the availability of highly effective, broad-spectrum agents, there remains a need for safer, more convenient and more environmentally friendly products that will overcome the ever-present threat of resistance development.The very high cost of discoveringand developinga new drug, especiallyfor usein livestock, is re%x%ed in the limited numberof new classes of antiparasiticagent launchedon the market. New strategiesare b&g &@d to
minim&e the cost of discovering potential drug candidates by maximising the chance of identifying a usefit target mechanismof action and by speedingthe time to discover and optimisea lead structure. Thesereiy heavily on new
technologies in target identification, screen development and lead optimisation. Examples of these will be discussed and speculationmade about the possiblefactors that could influence the future shapeof antiparasitic control. ,iT 1998 Australian Society for Parasitology.Publishedby ElsevierScienceLtd. All rights reserved. Kqwovds:
Market; Drug discovery; Screen; Target compound; Source library; Automation mechanism ---.--__
1. Intrddon
The effective control of helminth, insect and acarine parasites remains an important component of modern farming practices and of the care of household pets. Even sub-clinical infections or infestations can cause significant production losses and severe cases can result in death of the animal. In the home, control of flea and nematode infections in cats and dogs is important, both for the well-being of the animal and also to reduce the risk of zoonotic diseases to owners. The markets for antiparasitic products are among the largest segments of both
*Tel: 44 1304 646865; Fax: 44 1304 656595; e-mail: [email protected].
the livestock and companion-animal markets for healthcare agents (see Table 1) [I]. These market segments have expanded considerably in recent years. The livestock market increased following the introduction of the avermectin and milbemycin endectocides in which premium pricing was achieved on the basis of convenient, long-lasting, broad-spectrum parasite control. More recently, the companion-animal market has grown considerably due to the. sale through ethical channels of highly effective monthly treatments for fleas that have displaced 3esseffective over-the-counter (OTC) sprays and shampoos. Despite the availability of these highly ef%ctive and broad-spectrum agents, there remains a need for safer, more convenient and more environmentally friendly products that will overcome the ever-present threat of resistance development. However, rather than searching for new types of
0020-7519/99/s - see front matter IT) 1998 Australian Society for Parasitology. Published by Elsevier Science Ltd. All rights reser\ rd. Pff:SOOZO-7519(98)00193-3
96
M.J.
Witty
J International
Journalfor
Table 1 World livestock and companion-animal
Parasitology
95-103
product sales. 1996
Livestock
Parasiticides Performance enhancers Antimicrobials Other pharmaceuticals Biologicals Medicinal feed additives Nutritional feed additives Total (million USS)
29 (1999)
Companion animals
(million USS)
(%)
(million US$)
1465 310 1705 795 1585 2060 3530 11450
13 3 15 7 14 18 30 100
1295 355 585 610 65 490 3400
(%I 39 10 17 18 2 14 100
Sales prices ex manufacturers, compiled from Wood Mackenzie reports.
antiparasitic agent, the efforts of many animal health pharmaceutical companies have been focused on identifying agents from existing classes or developing new formulations of existing agents.
Relatively few classes of agent make up the major part of the market and introductions tend to come from the same class (Table 2). This paper will discuss the reasons behind this focus and explore alter-
Table 2 Animal health antiparasitic introductions 1996 sales (million US$)
First member
Launch date
No. of representatives
Spectrum
Sites of action
Organophosphates/ carbamates
260
Malathion
1952”
>30
Insects, acari, nematodes
Acetylcholinesterase inhibitors
Benzimidazoles
365
Carbaryl Thiabendazole
1957” 1961”
>12
Imidazothiazoles/ 260 tetrahydropyrimidines Benzoyl ureas (lufenuron)b 240
Tetramisole/ Pyrantel Diflubenzuron
1966” 1966 1972
Nematodes, tapeworms Nematodes
Light-stable pyrethroids Avermectins and milbemycins Imino imidazoline
200 995
Permethrin Ivermectin
1976
>I0
1981
6
100
Imadocloprid
1996
1
Phenyl pyrazole
80
Fipronil
1996
1
All other antiparasitics Total’
285 2785
Class
3 4
Tubulin polymerisation Acetylcholine receptor Insects (fleas), acari Chitin synthetase inhibitors Insects, acari Neurotoxins Nematodes, insects. Chloride channels acari (glutamate) Insects (fleas) Acetylcholine receptor Insects, acari Chloride channels (GABA)
a Date of first scientific publication. Milbemycin patented 1975, first avermectin scientific publication 1979. Including those for crops, over 100 pesticidal organophosphates have been made since 1938; similarly over 20 carbamates have appeared since 1951. bAt least nine benzoyl ureas have been commercialised for animal, public health and crop use. Sales figures are for lufenuron alone. ‘Total antiparasitic market 1996 (ex manufacturer) is USS2785miIlion (sales figures compiled from Wood Mackenzie report, July 1997).
hi.J.
Witty
/ international
Journal
native strategies that are being adopted to discover new classes of antiparasitic agents [2].
2. The opportunity
The antiparasitic market is fragmented by both host animal and target parasite species. Cattle represent the largest segment, but cats/dogs and sheep represent significant markets, with sales for horses and swine somewhat smaller. North America and Europe are the major markets, with Latin America and East Asia (including Australasia) close behind (Table 3). However, there is considerable regional variation in both the species and type of parasite that cause the most damage, even within host species (Table 4). Of the total US$3 billion plus market, significant sales are available only to agents that provide the opportunity for broad-spectrum parasite control across a range of species, with possibilities for administration that are applicable to differing worldwide management practices. The cost of discovering and developing a new animal drug is considerable. A 1996 Animal Pharm made an estimate of US$57million. However, for a new class of agent for food-animal use, it would now be much higher. A total cost before launch of over US$I 00 million would not be unrealistic, with a timeline from idea to launch of around 10 years. These figures are split almost equally with one-half on discovery and early development and the second half on full development. Clearly, the time and costs for discovery are reduced considerably if an agent from an existing class is being sought, and even
Table 3 Antiparasitic sales by region-1996 - -.__---__Region million US$
%
North America Latin America West Europe East Europe East Asia Rest of world Total
32 18 28 4 15 3 100
880 500 791 105 418 91 2185
for Parasitology
29
(19991
95-103
greater savings can be made by reformulation of generic agents. To obtain the pay-back on such major investments, the market opportunity clearly needs to be very significant. This has inevitably focused most of the major pharmaceutical companies on broadspectrum agents from existing classes. Few companies are making the long-term financial commitment to support the discovery of novel antiparasitic agents. Many companies rely on tinding new agents as spin-offs from larger crop-protection programmes, although the properties of such molecules may not be the same as those desired for a drug for use in animals on the farm or in the home. However, there are advantages in bringing a new compound class to the market. Firstly, a class of agent with a new mechanism of action will overcome the emergence of resistance to existing classes. A new spectrum of activity, duration of action, route of administration, improved environmental or target animal safety can provide a differentiating feature that will break livestock management paradigms and add market value. This differentiation will provide a greater opportunity for seizing market share than an agent from an existing class with marginal differentiating features. Secandly, a new class of agent will have a stronger proprietary position, with the advantages of being first to the market bringing a longer patent life and the opportunity to limit the competition by broad patent coverage. Clearly, there is a major risk/benefit balance to be achieved. To make the search for new agents an attractive commercial proposition, strategies are needed to reduce the cost and to increase the chance of success of the discovery phase.
3. The discovery process
A simplified process for the discovery of potential antiparasitic agents is shown in Fig. 1. In order to achieve one drug candidate with a promising profile, tens of compounds need to be tested in the target animal to identify the one with the ,appropriate safety and efficacy profile. To sebct these potential candidates. hundreds will have been tested
98
M.J.
Witty
1 International
Journalfor
Parasitology
29 (1999)
95-103
Table 4 Major parasites by region Region
Cattle
W Europe
GI nematodes
Sheep (Ostertagill)
Lungworm (Dictyocuutus) Biting lice (Bouicola) Ticks (Zxodes) N America
Latin America
E Europe
E Asia (incl. Australasia)
GI nematodes (Oster~crgia, but Haemonchus in South) Hornfly (Haematobiu) Ticks (Dermacentor, Amblyomma) Biting lice (Bouicola) Ticks (Boophifus)
Sheep scab (Psoroptes)
Fleas (Ctenoeeplalides)
GI nematodes Blowfly (Luciliu) Fluke (Fasciola) “GI nematodes”
GI nematodes Tapeworms Mange mites
Heartworm
(Dirofilari)
Ticks “Fleas, ticks, mange, worms”
GI nematodes
GI nematodes Ticks Warbles (Hypodermu) Screwworms (Callitroga) Mange mites
Sheep scab (Psoroptes)
GI nematodes
GI nematodes
Ticks Warbles (Hypodermu)
Lungwonns Mange Ticks
GI nematodes
GI nematodes (Haemonchus Ostertagia) Blowfly (Luciliu, Chrysomia)
Ticks (Boophilus)
Fleas (Ctenocephatiides)
Lice Flukes
“Fleas, ticks, mange, worms”
and
Fleas (Ctenocephdides) Heartworm
(Diro&rkz)
GI nematodes Mange Ticks
Major parasites are shown in bold. There are, however, huge variations in local parasites according to climatic variations in the different regions. GI = gastrointestinal.
in laboratory animals to confirm their quality-lead antiparasitic potential in vivo. These will arise, in turn, by screening thousands of compounds in in vitro screens to identify potent, broad-spectrum leads. Often these will have been selected following testing of many hundreds of thousands of compounds in different mechanistic screens. 3.1. Screen selection
Testing the antiparasitic efficacy of a compound in the target animal or laboratory animal against the real or a model infection will clearly identify a potential candidate. However, such compounds are few and far between, and compound- and resourceintensive in vivo models are used only in the late stage of the discovery process, once a lead series has been identified. The easiest way to discover an
antiparasitic lead is to apply it directly to the parasite in some way and to see if the parasite dies. For many years this has been the mainstay of antiparasitic screening. However, it does have drawbacks in the identification of early leads. Firstly, it is often not possible to use the target parasite due to difficulties in obtaining or culturing the appropriate species in vitro. Secondly, the in-vitro environment may be very different to the in-vivo one-this can result in more stress or less stress on the parasite depending on the mode of action of the test compound, which in turn can result in false positives and negatives. Thirdly, the drug still has to penetrate the parasite and reach its site of action before being metabolised. Although these barriers are not as great as those provided by the mammalian host in vivo, they can result in many intrinsically potent compounds being missed. Fourthly,
M.J. Wit+ i Internutionuf Journuffor Parasifofogy 29 (I9991 95-103
Fig. I Steps in the process of discovery and development for antiparasitics.
it limits the mode-of-action to those classes causing paralysis or death. These are often compounds that are broadly toxic which provide little therapeutic index. Increasingly, mechanistic screens are used in the early part of the drug-discovery process. They can be based on receptors, enzymes, ion channels, etc., and have the potential to overcome many of the problems seen with whole-organism screens. However, it is only with the advent of genomics and molecular biology that it has been possible to overcome problems of parasite material supply. Expressing parasite gene products in mammalian, yeast, bacterial or insect cell lines can be used to build screens that overcome the issues around supply of purified parasite receptor or enzyme material. In addition, the use of gene knock-out technology can provide early indications of target validation, through assessment of lethality. The increasing understanding of the genome of Cuenorhabditis elegans and the function of individual genes will help provide further targets for exploitation. Comparison of mammalian versus parasite material can also be used to provide an early indication of selective toxicity.
3.2. Screening The availability of expressed parasite material for mechanistic screens coupled with advanced robotics and miniaturisation, and computer controlled assay and analysis has transformed drug screening. No longer are screens measu.red in tens or hundreds of compounds per week, but in hundreds of thousands per week. Radioh ELBA, scintillation proximity, homogeneous timeresolved fluoresence, cellular reporter gene assays, etc., provide automatable endpoints. that allow potent actives to be readily identified. %nety-sixwell plate technologies are now being upgraded to 384- or even 1536-well plates to allow even more compound, reagent and time savings, and increased throughput. Plates of 9600 webs using nano-technology are being investigated [3-S]. Previously, mechanistic assays were developed into screens following validation ofthe &~gt-$t wing existing pharmacological standurds. This &ends to drive target selection towards those mechanisms of action associated with existing agents. The time and cost-savings (Table 5) associated with miniaturisation and automation of screening have
100
M.J.
Table 5 Example of costs of high-throughput pounds
Plates Peptide reagent Enzyme production Time (screening) Total Savings/assay Savings on 32 assays
Witty
screening
1 International
of 1 million
Journalfor
com-
96-well (singlet)
9600-well (triplicate)
us$4000 USS250 000 us 150 000 US$30 000 US$209 000 US$156000 US$5 000 000
US$200 US$150 umo 000 USS2500 US$53 000
allowed the validation and screening steps to be reversed [6-91. The leads generated from a highthroughput screen are themselves used to validate the target. These assays are generally set up to have a relatively low hit rate (< 1%) so that only the most intrinsically potent compounds are detected (< 1 PM). If no potent leads are identified, then the scientist now moves on to the next potential target. Identifying new targets and assays is becoming the rate-limiting step in drug discovery. 3.3. Compound
source
To identify the few quality antiparasitic lead compounds, many millions of compounds will have been tested. Where do these compounds come from? Initially, it is almost a random numbers game with odds improved by increasing the structural diversity of the compound supply. Later in the process,however, each compound will need to be individually designed and synthesised by a medicinal chemist to overcome potential weaknessesin the lead compound, e.g., potency, metabolic vulnerability, distribution, etc. A highly successful source of compounds has been from natural-product extracts (Fig. 2). These represent a highly structurally diverse range of compounds [lo]. However, many of the more common organisms have been heavily plundered by screening over the years and, unless effective dereplication processesare in place, much time can be wasted following up weedsand known compounds. Mechanistic screens considerably reduce these
Parasitology
29 (1999)
95-103
difficulties by eliminating biocides. The focus is now on testing extracts from more exotic micro-organisms and higher organisms, e.g., plants, seacreatures, etc. However, these bring their own problems once a hit is identified and scale-up is needed. A company’s compound files represent one of their major assets.The hundreds of thousands of compounds stored in the company files reflect all the compounds that have been made in previous drug-discovery programmes. However, these have often already been screenedwidely and reflect relatively limited structural diversity. New sources of compounds are being widely sought by all companies. Compound libraries, containing millions of individual pure compounds or mixtures, are now being prepared by automation and combinatorial synthesis (Fig. 3) [ll-141. Simultaneous reaction of a number of substrates with a number of reagents can produce mixtures of compounds that can be screened directly. This process can be used to rapidly generate libraries of many thousands of compounds, greatly increasing the diversity of the compound files. Although isolating the active component in a mixture can take some time, further innovative technologies are being introduced to streamline this process also. While such combinatorial libraries will increase the source of compounds for testing into the millions, this falls short of the hundreds of millions of possible “drug-sized” compounds that could be made. When the target receptor or enzyme structure is known and can be modelled on a computer [15], then a virtual library of compounds can be screened against the model to identify structural classesfor closer attention. Such approaches require only faster computers and detailed knowledge of the targets. 3.4. Leadfollow-up
and optimisation
Once a potent mechanistic active is identified, then it is up to the medicinal chemist to modify its structure to provide a compound that overcomes the deficiencies of the lead. The processhaschanged relatively little over recent years. However, a number of new tools are available to streamline the
M.J.
Witty
/ International
Journalfor
Parasitology
29 (1999)
95-103
101
PF-1022A
Paraherquamide
OH Ryamdine Avermeclln
Bla
Fig. 2. Natural
product-derived
process. Computer modelling can be used to assist the design of compounds that will fit the receptor better, when a model of the enzyme or active site is known. In-vitro metabolic assays can be used to identify the susceptibility of a molecule to metabolism and HPLC-mass spectrometry to identify the metabolites and site of metabolism. In-vitro cell
antiparasitic
agents
layers can be used to assess penetration across tissue compartments. Even the synthesis process itself has changed. In addition to chemistry advances, robotits and automation can be used to speed up synthesis, often running many similar reactions in parallel and using autopurification so that many thousands of analogues can be generated in a short
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time. All of these processes are aimed at shortening the time and cost of the drug-discovery process.
4. The future There is clearly a great opportunity for academic and commercial researchers to work together to identify new antiparasitic agents. Increasing our understanding of the biology of parasites as well as the epidemiology of the diseases will be key components of that. Will these advances be sufficient to overcome the high risk of novel antiparasitic-agent discovery? We shall not know the answer for some time yet. Ideas being progressed in the discovery phase now are unlikely to hit the market until 2005 or beyond. What will the market be like by then? Will resistance be rampant? Will generic broadspectrum antiparasitics have devalued the market opportunity? Will the “green” lobby have won the day and any drug for use in food animals be prohibited? Time will tell. What are the alternatives? Will vaccines that have
Parasitology
29 (1999)
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been promised for so long finally be realised? Will transgenic animals with intrinsic resistance to parasites be available? Will biological control measures be the order of the day? These are important questions for those involved in drug discovery.
Acknowledgements The author gratefully acknowledges the support of Brian Manger, Pfizer Central Research, for his assistance in assembling the market data, provided courtesy of Wood Mackenzie.
References [l]
Allen H. Antiparasitics: products and markets. London: PJB Publications, 1995. [2] Schmatz DM, Schaeffer JM. Antiparasitic agents. In: Bristol JA, editor. Annual reports in medicinal chemistry. San Diego: Academic Press, 1991;161-70. [3] Burbaum JJ, Sigal NH. New technologies for high-throughput screening. Curr Opin Chem Biol 1997; 1:72-78.
M.J.
Witfv
/ International
Journalfor
[4] Houston JG, Banks M. The chemical-biological interface: developments in automated and miniaturised screening technologies. Curr Opin Biotechnol 1997;8:734-740. [5] Harrison W. Changes in scale in automated pharmaceutical research. Drug Discov Today 1998;3:343-349. [6] Burbaum JJ. Miniaturisation technologies in HTS: how fast, how small. how soon? Drug Discov Today 1998;3:313322. [7] Major J. Challenges and opportunities in HTS: implications for new technologies. J Biomol Screen 1998;3: 13-l 7. [8] Oldenburg KR. Microtechnologies and miniaturisation: tools, techniques and novel applications for the pharmaceutical industry. Conference Abstracts, Berlin. 889 December 1997. [9] Oldenburg KR, Zhang JH. Chem JH, et al. Assay miniaturization for ultra-high throughput screening of combinatorial and discrete compound libraries: a 9600-well assay system. J Biomol Screen 1998;3:55-62.
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29 (1999)
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103
[IO] Londerhausen M. Approaches to new parasiticides. Pestic Sci 1996:48:269-292. [II] Lebl M, Leblova Z. Dynamic database of references in molecular diversity. Internet http://wwwSz.com. [12] Terrett NK, Gardner M, Gordon DW, Kobylecki RJ, Steele J. Combinatorial synthesis: the design of compound libraries and their application to drug discovery. Tetrahedron 1995;51:8135-8173. [13] Gordon EM. Barrett RW, Dower WJ, Fodor SPA, Gallop MA. Applications of combinatorial technologies to drug discovery 2. Combinatorial organic synthesis, library screening strategies, and future directions. J Med Chem 1994;37:1385-1401. [14] Fruchtel JS, Jung G. Organic chemistry on solid supports. Angew Chem Int Ed 1996;35:17-42. [I 51 Broughton HB. Molecular modeling. Curr Opin Chem Biol 1997;1:392-398.
International Journal for Parasitology 29 ( 1999) 95- IO3
Current strategies in the search for novel antiparasitic
agents
Michael J. Witty* Animal
Health
Discot>ery,
Pfizer
Central
Research,
Ramsgate
Road, Sandwich,
Kent CT13 9NJ.
UK
Received 24 March 1998; accepted 7 October 1998
Abstract
The market for antiparasitic products comprises the largest segment for sales of livestock and companion-animal healthcare agents. Despite the availability of highly effective, broad-spectrum agents, there remains a need for safer, more convenient and more environmentally friendly products that will overcome the ever-present threat of resistance development.The very high cost of discoveringand developinga new drug, especiallyfor usein livestock, is re%x%ed in the limited numberof new classes of antiparasiticagent launchedon the market. New strategiesare b&g &@d to
minim&e the cost of discovering potential drug candidates by maximising the chance of identifying a usefit target mechanismof action and by speedingthe time to discover and optimisea lead structure. Thesereiy heavily on new
technologies in target identification, screen development and lead optimisation. Examples of these will be discussed and speculationmade about the possiblefactors that could influence the future shapeof antiparasitic control. ,iT 1998 Australian Society for Parasitology.Publishedby ElsevierScienceLtd. All rights reserved. Kqwovds:
Market; Drug discovery; Screen; Target compound; Source library; Automation mechanism ---.--__
1. Intrddon
The effective control of helminth, insect and acarine parasites remains an important component of modern farming practices and of the care of household pets. Even sub-clinical infections or infestations can cause significant production losses and severe cases can result in death of the animal. In the home, control of flea and nematode infections in cats and dogs is important, both for the well-being of the animal and also to reduce the risk of zoonotic diseases to owners. The markets for antiparasitic products are among the largest segments of both
*Tel: 44 1304 646865; Fax: 44 1304 656595; e-mail: [email protected].
the livestock and companion-animal markets for healthcare agents (see Table 1) [I]. These market segments have expanded considerably in recent years. The livestock market increased following the introduction of the avermectin and milbemycin endectocides in which premium pricing was achieved on the basis of convenient, long-lasting, broad-spectrum parasite control. More recently, the companion-animal market has grown considerably due to the. sale through ethical channels of highly effective monthly treatments for fleas that have displaced 3esseffective over-the-counter (OTC) sprays and shampoos. Despite the availability of these highly ef%ctive and broad-spectrum agents, there remains a need for safer, more convenient and more environmentally friendly products that will overcome the ever-present threat of resistance development. However, rather than searching for new types of
0020-7519/99/s - see front matter IT) 1998 Australian Society for Parasitology. Published by Elsevier Science Ltd. All rights reser\ rd. Pff:SOOZO-7519(98)00193-3
96
M.J.
Witty
J International
Journalfor
Table 1 World livestock and companion-animal
Parasitology
95-103
product sales. 1996
Livestock
Parasiticides Performance enhancers Antimicrobials Other pharmaceuticals Biologicals Medicinal feed additives Nutritional feed additives Total (million USS)
29 (1999)
Companion animals
(million USS)
(%)
(million US$)
1465 310 1705 795 1585 2060 3530 11450
13 3 15 7 14 18 30 100
1295 355 585 610 65 490 3400
(%I 39 10 17 18 2 14 100
Sales prices ex manufacturers, compiled from Wood Mackenzie reports.
antiparasitic agent, the efforts of many animal health pharmaceutical companies have been focused on identifying agents from existing classes or developing new formulations of existing agents.
Relatively few classes of agent make up the major part of the market and introductions tend to come from the same class (Table 2). This paper will discuss the reasons behind this focus and explore alter-
Table 2 Animal health antiparasitic introductions 1996 sales (million US$)
First member
Launch date
No. of representatives
Spectrum
Sites of action
Organophosphates/ carbamates
260
Malathion
1952”
>30
Insects, acari, nematodes
Acetylcholinesterase inhibitors
Benzimidazoles
365
Carbaryl Thiabendazole
1957” 1961”
>12
Imidazothiazoles/ 260 tetrahydropyrimidines Benzoyl ureas (lufenuron)b 240
Tetramisole/ Pyrantel Diflubenzuron
1966” 1966 1972
Nematodes, tapeworms Nematodes
Light-stable pyrethroids Avermectins and milbemycins Imino imidazoline
200 995
Permethrin Ivermectin
1976
>I0
1981
6
100
Imadocloprid
1996
1
Phenyl pyrazole
80
Fipronil
1996
1
All other antiparasitics Total’
285 2785
Class
3 4
Tubulin polymerisation Acetylcholine receptor Insects (fleas), acari Chitin synthetase inhibitors Insects, acari Neurotoxins Nematodes, insects. Chloride channels acari (glutamate) Insects (fleas) Acetylcholine receptor Insects, acari Chloride channels (GABA)
a Date of first scientific publication. Milbemycin patented 1975, first avermectin scientific publication 1979. Including those for crops, over 100 pesticidal organophosphates have been made since 1938; similarly over 20 carbamates have appeared since 1951. bAt least nine benzoyl ureas have been commercialised for animal, public health and crop use. Sales figures are for lufenuron alone. ‘Total antiparasitic market 1996 (ex manufacturer) is USS2785miIlion (sales figures compiled from Wood Mackenzie report, July 1997).
hi.J.
Witty
/ international
Journal
native strategies that are being adopted to discover new classes of antiparasitic agents [2].
2. The opportunity
The antiparasitic market is fragmented by both host animal and target parasite species. Cattle represent the largest segment, but cats/dogs and sheep represent significant markets, with sales for horses and swine somewhat smaller. North America and Europe are the major markets, with Latin America and East Asia (including Australasia) close behind (Table 3). However, there is considerable regional variation in both the species and type of parasite that cause the most damage, even within host species (Table 4). Of the total US$3 billion plus market, significant sales are available only to agents that provide the opportunity for broad-spectrum parasite control across a range of species, with possibilities for administration that are applicable to differing worldwide management practices. The cost of discovering and developing a new animal drug is considerable. A 1996 Animal Pharm made an estimate of US$57million. However, for a new class of agent for food-animal use, it would now be much higher. A total cost before launch of over US$I 00 million would not be unrealistic, with a timeline from idea to launch of around 10 years. These figures are split almost equally with one-half on discovery and early development and the second half on full development. Clearly, the time and costs for discovery are reduced considerably if an agent from an existing class is being sought, and even
Table 3 Antiparasitic sales by region-1996 - -.__---__Region million US$
%
North America Latin America West Europe East Europe East Asia Rest of world Total
32 18 28 4 15 3 100
880 500 791 105 418 91 2185
for Parasitology
29
(19991
95-103
greater savings can be made by reformulation of generic agents. To obtain the pay-back on such major investments, the market opportunity clearly needs to be very significant. This has inevitably focused most of the major pharmaceutical companies on broadspectrum agents from existing classes. Few companies are making the long-term financial commitment to support the discovery of novel antiparasitic agents. Many companies rely on tinding new agents as spin-offs from larger crop-protection programmes, although the properties of such molecules may not be the same as those desired for a drug for use in animals on the farm or in the home. However, there are advantages in bringing a new compound class to the market. Firstly, a class of agent with a new mechanism of action will overcome the emergence of resistance to existing classes. A new spectrum of activity, duration of action, route of administration, improved environmental or target animal safety can provide a differentiating feature that will break livestock management paradigms and add market value. This differentiation will provide a greater opportunity for seizing market share than an agent from an existing class with marginal differentiating features. Secandly, a new class of agent will have a stronger proprietary position, with the advantages of being first to the market bringing a longer patent life and the opportunity to limit the competition by broad patent coverage. Clearly, there is a major risk/benefit balance to be achieved. To make the search for new agents an attractive commercial proposition, strategies are needed to reduce the cost and to increase the chance of success of the discovery phase.
3. The discovery process
A simplified process for the discovery of potential antiparasitic agents is shown in Fig. 1. In order to achieve one drug candidate with a promising profile, tens of compounds need to be tested in the target animal to identify the one with the ,appropriate safety and efficacy profile. To sebct these potential candidates. hundreds will have been tested
98
M.J.
Witty
1 International
Journalfor
Parasitology
29 (1999)
95-103
Table 4 Major parasites by region Region
Cattle
W Europe
GI nematodes
Sheep (Ostertagill)
Lungworm (Dictyocuutus) Biting lice (Bouicola) Ticks (Zxodes) N America
Latin America
E Europe
E Asia (incl. Australasia)
GI nematodes (Oster~crgia, but Haemonchus in South) Hornfly (Haematobiu) Ticks (Dermacentor, Amblyomma) Biting lice (Bouicola) Ticks (Boophifus)
Sheep scab (Psoroptes)
Fleas (Ctenoeeplalides)
GI nematodes Blowfly (Luciliu) Fluke (Fasciola) “GI nematodes”
GI nematodes Tapeworms Mange mites
Heartworm
(Dirofilari)
Ticks “Fleas, ticks, mange, worms”
GI nematodes
GI nematodes Ticks Warbles (Hypodermu) Screwworms (Callitroga) Mange mites
Sheep scab (Psoroptes)
GI nematodes
GI nematodes
Ticks Warbles (Hypodermu)
Lungwonns Mange Ticks
GI nematodes
GI nematodes (Haemonchus Ostertagia) Blowfly (Luciliu, Chrysomia)
Ticks (Boophilus)
Fleas (Ctenocephatiides)
Lice Flukes
“Fleas, ticks, mange, worms”
and
Fleas (Ctenocephdides) Heartworm
(Diro&rkz)
GI nematodes Mange Ticks
Major parasites are shown in bold. There are, however, huge variations in local parasites according to climatic variations in the different regions. GI = gastrointestinal.
in laboratory animals to confirm their quality-lead antiparasitic potential in vivo. These will arise, in turn, by screening thousands of compounds in in vitro screens to identify potent, broad-spectrum leads. Often these will have been selected following testing of many hundreds of thousands of compounds in different mechanistic screens. 3.1. Screen selection
Testing the antiparasitic efficacy of a compound in the target animal or laboratory animal against the real or a model infection will clearly identify a potential candidate. However, such compounds are few and far between, and compound- and resourceintensive in vivo models are used only in the late stage of the discovery process, once a lead series has been identified. The easiest way to discover an
antiparasitic lead is to apply it directly to the parasite in some way and to see if the parasite dies. For many years this has been the mainstay of antiparasitic screening. However, it does have drawbacks in the identification of early leads. Firstly, it is often not possible to use the target parasite due to difficulties in obtaining or culturing the appropriate species in vitro. Secondly, the in-vitro environment may be very different to the in-vivo one-this can result in more stress or less stress on the parasite depending on the mode of action of the test compound, which in turn can result in false positives and negatives. Thirdly, the drug still has to penetrate the parasite and reach its site of action before being metabolised. Although these barriers are not as great as those provided by the mammalian host in vivo, they can result in many intrinsically potent compounds being missed. Fourthly,
M.J. Wit+ i Internutionuf Journuffor Parasifofogy 29 (I9991 95-103
Fig. I Steps in the process of discovery and development for antiparasitics.
it limits the mode-of-action to those classes causing paralysis or death. These are often compounds that are broadly toxic which provide little therapeutic index. Increasingly, mechanistic screens are used in the early part of the drug-discovery process. They can be based on receptors, enzymes, ion channels, etc., and have the potential to overcome many of the problems seen with whole-organism screens. However, it is only with the advent of genomics and molecular biology that it has been possible to overcome problems of parasite material supply. Expressing parasite gene products in mammalian, yeast, bacterial or insect cell lines can be used to build screens that overcome the issues around supply of purified parasite receptor or enzyme material. In addition, the use of gene knock-out technology can provide early indications of target validation, through assessment of lethality. The increasing understanding of the genome of Cuenorhabditis elegans and the function of individual genes will help provide further targets for exploitation. Comparison of mammalian versus parasite material can also be used to provide an early indication of selective toxicity.
3.2. Screening The availability of expressed parasite material for mechanistic screens coupled with advanced robotics and miniaturisation, and computer controlled assay and analysis has transformed drug screening. No longer are screens measu.red in tens or hundreds of compounds per week, but in hundreds of thousands per week. Radioh ELBA, scintillation proximity, homogeneous timeresolved fluoresence, cellular reporter gene assays, etc., provide automatable endpoints. that allow potent actives to be readily identified. %nety-sixwell plate technologies are now being upgraded to 384- or even 1536-well plates to allow even more compound, reagent and time savings, and increased throughput. Plates of 9600 webs using nano-technology are being investigated [3-S]. Previously, mechanistic assays were developed into screens following validation ofthe &~gt-$t wing existing pharmacological standurds. This &ends to drive target selection towards those mechanisms of action associated with existing agents. The time and cost-savings (Table 5) associated with miniaturisation and automation of screening have
100
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Table 5 Example of costs of high-throughput pounds
Plates Peptide reagent Enzyme production Time (screening) Total Savings/assay Savings on 32 assays
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com-
96-well (singlet)
9600-well (triplicate)
us$4000 USS250 000 us 150 000 US$30 000 US$209 000 US$156000 US$5 000 000
US$200 US$150 umo 000 USS2500 US$53 000
allowed the validation and screening steps to be reversed [6-91. The leads generated from a highthroughput screen are themselves used to validate the target. These assays are generally set up to have a relatively low hit rate (< 1%) so that only the most intrinsically potent compounds are detected (< 1 PM). If no potent leads are identified, then the scientist now moves on to the next potential target. Identifying new targets and assays is becoming the rate-limiting step in drug discovery. 3.3. Compound
source
To identify the few quality antiparasitic lead compounds, many millions of compounds will have been tested. Where do these compounds come from? Initially, it is almost a random numbers game with odds improved by increasing the structural diversity of the compound supply. Later in the process,however, each compound will need to be individually designed and synthesised by a medicinal chemist to overcome potential weaknessesin the lead compound, e.g., potency, metabolic vulnerability, distribution, etc. A highly successful source of compounds has been from natural-product extracts (Fig. 2). These represent a highly structurally diverse range of compounds [lo]. However, many of the more common organisms have been heavily plundered by screening over the years and, unless effective dereplication processesare in place, much time can be wasted following up weedsand known compounds. Mechanistic screens considerably reduce these
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difficulties by eliminating biocides. The focus is now on testing extracts from more exotic micro-organisms and higher organisms, e.g., plants, seacreatures, etc. However, these bring their own problems once a hit is identified and scale-up is needed. A company’s compound files represent one of their major assets.The hundreds of thousands of compounds stored in the company files reflect all the compounds that have been made in previous drug-discovery programmes. However, these have often already been screenedwidely and reflect relatively limited structural diversity. New sources of compounds are being widely sought by all companies. Compound libraries, containing millions of individual pure compounds or mixtures, are now being prepared by automation and combinatorial synthesis (Fig. 3) [ll-141. Simultaneous reaction of a number of substrates with a number of reagents can produce mixtures of compounds that can be screened directly. This process can be used to rapidly generate libraries of many thousands of compounds, greatly increasing the diversity of the compound files. Although isolating the active component in a mixture can take some time, further innovative technologies are being introduced to streamline this process also. While such combinatorial libraries will increase the source of compounds for testing into the millions, this falls short of the hundreds of millions of possible “drug-sized” compounds that could be made. When the target receptor or enzyme structure is known and can be modelled on a computer [15], then a virtual library of compounds can be screened against the model to identify structural classesfor closer attention. Such approaches require only faster computers and detailed knowledge of the targets. 3.4. Leadfollow-up
and optimisation
Once a potent mechanistic active is identified, then it is up to the medicinal chemist to modify its structure to provide a compound that overcomes the deficiencies of the lead. The processhaschanged relatively little over recent years. However, a number of new tools are available to streamline the
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101
PF-1022A
Paraherquamide
OH Ryamdine Avermeclln
Bla
Fig. 2. Natural
product-derived
process. Computer modelling can be used to assist the design of compounds that will fit the receptor better, when a model of the enzyme or active site is known. In-vitro metabolic assays can be used to identify the susceptibility of a molecule to metabolism and HPLC-mass spectrometry to identify the metabolites and site of metabolism. In-vitro cell
antiparasitic
agents
layers can be used to assess penetration across tissue compartments. Even the synthesis process itself has changed. In addition to chemistry advances, robotits and automation can be used to speed up synthesis, often running many similar reactions in parallel and using autopurification so that many thousands of analogues can be generated in a short
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time. All of these processes are aimed at shortening the time and cost of the drug-discovery process.
4. The future There is clearly a great opportunity for academic and commercial researchers to work together to identify new antiparasitic agents. Increasing our understanding of the biology of parasites as well as the epidemiology of the diseases will be key components of that. Will these advances be sufficient to overcome the high risk of novel antiparasitic-agent discovery? We shall not know the answer for some time yet. Ideas being progressed in the discovery phase now are unlikely to hit the market until 2005 or beyond. What will the market be like by then? Will resistance be rampant? Will generic broadspectrum antiparasitics have devalued the market opportunity? Will the “green” lobby have won the day and any drug for use in food animals be prohibited? Time will tell. What are the alternatives? Will vaccines that have
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been promised for so long finally be realised? Will transgenic animals with intrinsic resistance to parasites be available? Will biological control measures be the order of the day? These are important questions for those involved in drug discovery.
Acknowledgements The author gratefully acknowledges the support of Brian Manger, Pfizer Central Research, for his assistance in assembling the market data, provided courtesy of Wood Mackenzie.
References [l]
Allen H. Antiparasitics: products and markets. London: PJB Publications, 1995. [2] Schmatz DM, Schaeffer JM. Antiparasitic agents. In: Bristol JA, editor. Annual reports in medicinal chemistry. San Diego: Academic Press, 1991;161-70. [3] Burbaum JJ, Sigal NH. New technologies for high-throughput screening. Curr Opin Chem Biol 1997; 1:72-78.
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[4] Houston JG, Banks M. The chemical-biological interface: developments in automated and miniaturised screening technologies. Curr Opin Biotechnol 1997;8:734-740. [5] Harrison W. Changes in scale in automated pharmaceutical research. Drug Discov Today 1998;3:343-349. [6] Burbaum JJ. Miniaturisation technologies in HTS: how fast, how small. how soon? Drug Discov Today 1998;3:313322. [7] Major J. Challenges and opportunities in HTS: implications for new technologies. J Biomol Screen 1998;3: 13-l 7. [8] Oldenburg KR. Microtechnologies and miniaturisation: tools, techniques and novel applications for the pharmaceutical industry. Conference Abstracts, Berlin. 889 December 1997. [9] Oldenburg KR, Zhang JH. Chem JH, et al. Assay miniaturization for ultra-high throughput screening of combinatorial and discrete compound libraries: a 9600-well assay system. J Biomol Screen 1998;3:55-62.
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[IO] Londerhausen M. Approaches to new parasiticides. Pestic Sci 1996:48:269-292. [II] Lebl M, Leblova Z. Dynamic database of references in molecular diversity. Internet http://wwwSz.com. [12] Terrett NK, Gardner M, Gordon DW, Kobylecki RJ, Steele J. Combinatorial synthesis: the design of compound libraries and their application to drug discovery. Tetrahedron 1995;51:8135-8173. [13] Gordon EM. Barrett RW, Dower WJ, Fodor SPA, Gallop MA. Applications of combinatorial technologies to drug discovery 2. Combinatorial organic synthesis, library screening strategies, and future directions. J Med Chem 1994;37:1385-1401. [14] Fruchtel JS, Jung G. Organic chemistry on solid supports. Angew Chem Int Ed 1996;35:17-42. [I 51 Broughton HB. Molecular modeling. Curr Opin Chem Biol 1997;1:392-398.