2020 Volume 43 Issue 2 Pages 216-220
Drugs are developed through basic studies and clinical trials. In basic studies, researchers seek drug candidates using in vitro evaluation systems and subsequently examine their effectiveness in animal experiments as in vivo evaluations. Drug candidates identified in basic studies are tested to determine whether they are effective against human diseases in clinical trials. However, most drug candidates identified in in vitro evaluation systems do not show therapeutic effects in animal experiments due to pharmacokinetics and toxicity problems in the in vivo evaluations. This review outlines drug discovery using insect disease models that allow us to perform in vivo screening. Since insects have various advantages as experimental animals such as low cost for rearing and few ethical concerns, researchers can perform large-scale in vivo screening to find drug candidates. Silkworms are insects frequently used for studies of drug efficacy, pharmacokinetics, and toxicity. Based on silkworm research, I describe the benefits of using insect disease models for drug discovery. The use of insect disease models for in vivo screening is expected to facilitate drug discovery.
The drug discovery process is broadly divided into basic studies and clinical trials (Fig. 1). In basic studies, researchers seek drug candidates that exert beneficial effects in cultured cells and activity on target proteins as shown by large-scale screening in in vitro experimental systems.1,2) When drug candidates are identified, they are optimized by chemical modification and formulation.3–5) Next, preclinical tests using experimental animals are performed. Clinical trials confirm whether the drug candidates identified in basic studies are effective in humans.
Human disease models using insects can be included in the basic study phase. With the addition of insect disease models, all basic studies for drug discovery will be performed as in vivo experiments. (Color figure can be accessed in the online version.)
Unfortunately, research on most drug candidates identified in basic studies is discontinued in the preclinical and clinical research stages. The pharmacokinetics and toxicity (ADMET: absorption, distribution, metabolism, excretion, and toxicity) of drug candidates are often problematic. The problems occur when a sufficient amount of the drug candidate does not reach the site of action in animals owing to poor pharmacokinetics or when it is too toxic and cannot be administered until a therapeutically effective concentration is reached. To solve these ADMET problems by optimization of drug candidates through chemical modification and formulation, animal experiments are necessary to test effectiveness. However, the use of large numbers of mammals, such as mice and rats, causes cost and ethical problems. Recently, the focus has been on insects as useful animals in the initial stage of basic study to seek drug candidates that show therapeutic effects6–9) (Fig. 1).
In the 3Rs, an international principle of experiments using mammals, the use of alternative animals (replacement), pain relief (refinement), and reduction of the number of animals used (reduction) are proposed.10) Experiments with invertebrates including insects are consistent with the concept of replacement. Invertebrates generally have the following advantages compared with mammals: 1) lower breeding costs; 2) more individuals can be reared in smaller spaces; 3) fewer ethical problems when euthanized; and 4) smaller tissue samples needed due to smaller body sizes (Table 1). Attempts have been made to use invertebrates such as nematodes (Caenorhabditis elegans), fruit flies (Drosophila melanogaster), greater wax moths (Galleria mellonella), and silkworms (Bombyx mori) as models for drug discovery research.6–9,11–14) Nematodes and fruit flies have advantages in genetic research, but because of their small size it is difficult to perform quantitative administration of sample solutions into their blood and perform biochemical experiments that require insect blood samples (Table 1). Therefore, there has been little research on the screening and optimization of compounds based on the evaluation of therapeutic effects by injection.
Animal | Cost for rearing | Space for rearing | Approval by an ethics committee | Potential for escape requiring biosafety measures | Quantitative injection of samples via syringe | Reference |
---|---|---|---|---|---|---|
Silkworm (larva) [Bombyx mori] | Low | Small | Not necessary | Low | Easy | 16 |
Fruit fly (adult) [Drosophila melanogaster] | Low | Small | Not necessary | High | Difficult | 54 |
Nematode [Caenorhabditis elegans] | Low | Small | Not necessary | Low | Difficult | 55 |
Greater wax moth (larva) [Galleria mellonella] | Low | Small | Not necessary | Low | Easy | 56 |
Mouse [Mus musculus] | High | Large | Necessary | High | Easy | 57–59 |
Adapted from Ishii et al.33)
On the other hand, silkworms have the advantages that quantitative injection of sample solutions using syringes and determination of enzyme activity in the blood can be easily performed.9) Furthermore, in silkworm experiments, researchers can distinguish between intrahemolymph injection and intramidgut injection.9,15–17) The former corresponds to intravenous injection in humans, and the latter to oral administration. The injection techniques are important for the evaluation of pharmacokinetics and sample toxicity. These advantages of silkworms allow their use in large-scale in vivo screening and optimization through monitoring of the therapeutic activities of drug candidates (Fig. 1).
Silkworms are useful for evaluating the pharmacokinetics and toxicity of compounds based on the advantages described above. In mammals, organs such as the intestinal tract, liver, and kidney govern drug pharmacokinetics. Recent research has revealed that silkworms also have functionally similar organs that affect drug pharmacokinetics and toxicity.18,19) Many in vitro and in vivo analyses revealed that the absorption of compounds from the intestinal tract of silkworms is similar to that of mammals.15–17,20) The total clearance, volume of distribution, and half-life values of antimicrobial agents such as chloramphenicol, tetracycline, vancomycin, rifampicin, micafungin, and fluconazole differ by less than 10-fold between silkworms and mammals.21) Toxicity to animals is quantitatively indicated by the LD50 value, the dose of a compound required to kill half of an experimental group. LD50 values of most compounds in silkworms and mammals are similar.15,22) Tissue damage by toxic substances can be measured based on alanine aminotransferase (ALT) activity in animal serum, because ALT is an enzyme eluted from damaged tissue. Carbon tetrachloride is a toxic compound activated by an oxidative reaction in the liver. The administration of carbon tetrachloride to silkworms led to increased ALT activity in the silkworm hemolymph.18,23) The increase in ALT activity in the hemolymph of silkworms after carbon tetrachloride administration was inhibited by the antioxidant N-acetyl cysteine.23) Those results suggested that toxic substances can be evaluated by the oxidation reaction of CYP in silkworms. Moreover, silkworms excrete several compounds from the hemolymph into the intestine through a conjugation reaction after metabolism via CYP.24) Those findings imply that the pharmacokinetics and toxicity of compounds in silkworms are similar to those in mammals. It is assumed that other insects, such as fruit flies, may also have similar systems. Therefore, insects are useful for evaluating drug efficacy in terms of pharmacokinetics and toxicity.
The development of treatments for infectious diseases, cancer, and lifestyle-related diseases is an important goal, and animal disease models are needed to develop therapeutic drugs. Many infectious disease models using insects such as nematodes, fruit flies, greater wax moths, and silkworms have been established.9,13,14,17,25–36) These infection models are based on the death of insects with the administration of pathogenic microorganisms. Large-scale in vivo screening has identified virulence genes of pathogens and defense-related genes of hosts. As a representative example, the Toll receptor was discovered using a Drosophila fungal infection model.37) Toll-like receptors, which are homologues of the Toll receptor, play important roles in the mammalian innate immune response, and thus the significance of basic medical research using insects was established.38,39) Moreover, Drosophila thyroid cancer models and mutants exhibiting Parkinson’s disease-like symptoms in silkworms were reported.40–43) Bioresources of gene mutants of insects have been prepared, and various disease models using those mutants will be established in the future. Furthermore, silkworms become diabetic with the intake of a high-sugar diet.44–46) The development of insect models of lifestyle-related diseases caused by excessive nutrition is also expected. Disease models using insects can be utilized not only in basic medical research but also in the drug discovery process.
One of the landmarks of drug discovery research using insects was the contribution to the development of vandetanib, a chemotherapeutic agent used to treat thyroid cancer patients.41) Since many anticancer agents are toxic to healthy cells, it is important to evaluate toxicity in animals. A thyroid cancer model using Drosophila genetically mutated for the development of molecular targeted drugs contributed to the identification of candidate compounds.40,47) Vandetanib was approved by the U.S. Food and Drug Administration and became one of the chemotherapeutic agents used to treat patients with thyroid cancer.48) Therefore, human disease models using insects can contribute to drug discovery for cancer therapy.
Insects are also used in the development of agents to treat infectious diseases, and useful compounds have been obtained through large-scale in vivo screening. The administration of antimicrobial drugs showed therapeutic effects in silkworms infected with pathogens.15,29) Lysocin E, nosokomycin, ASP2397, and GPI0363 were identified by exploratory research using a silkworm infection model from microbial culture broths and chemical libraries as novel antimicrobial compounds that showed therapeutic effects in mouse infection experiments.49–52) Whether these compounds will be approved after clinical trials is a question for the future.
Diabetes is caused by toxicity due to postprandial hyperglycemia, and agents suppressing postprandial hyperglycemia are effective in the prevention and treatment of diabetes. In vivo screening using a postprandial hyperglycemia model in silkworms identified Enterococcus faecalis YM0831 as a functional lactic acid bacterium that suppresses postprandial hyperglycemia.53)E. faecalis YM0831 also inhibited postprandial hyperglycemia in humans. Therefore, substances beneficial to human health can be obtained using human disease silkworm models.
In conclusion, human disease models using insects are useful for identifying drug candidates in large-scale in vivo screening (Fig. 1). Further research on the pharmacokinetics and toxicity of compounds will help us to understand the advantages of using insect models of human disease. Therefore, researchers should continue and expand the use of disease models for drug discovery at the basic study stage.
The author declares no conflict of interest.