Production of domestic redback spider antitoxin and preclinical studies

Production of domestic redback spider antitoxin and preclinical studies

Department of Safety and Laboratory Medicine, National Institute of Infectious Diseases (NIID)

1. Introduction

The redback spider (Latrodectus hasseltii: Fig. 1) is a small, female-only poisonous species of spider that is classified in the family Diptera. Its Japanese name means “”red-backed spider,”” and it is widely distributed from East Asia to Australia and South Pacific countries. It was previously not found in Japan, but since its discovery in September 1995 in Takaishi City, Osaka Prefecture, and then in a reclaimed land in Yokkaichi City, Mie Prefecture, it has expanded its habitat area nationwide, and as of August 2019, it is distributed throughout Japan except in Akita and Aomori Prefectures (Fig. 2: ref. 1). It is a living organism. It is not aggressive, but it lives in close proximity to human habitats. It nests and breeds in sunny, warm places where there are insects to feed, such as inside gutters, in the crevices of their metal lids, inside drainpipes on residential lots, at the base of fences, and in blocks in flower beds. Immediately after being bitten, the bite causes only mild pain, but the pain gradually increases, and abdominal and chest pain may occur. Although rare, severe cases cause vomiting, fever, high blood pressure, tachycardia, and other systemic symptoms caused by neurotoxins. In Australia, where the disease originated, there have been reports of deaths, and antitoxin treatment is also provided for severe cases (Refs. 2-4). There have been 96 published cases of bites in Osaka Prefecture, where domestic survey data is available, from 1995 to 2019, with a clear increase from 2006 until around 2013, mainly in Osaka Prefecture, and the situation has leveled off since then (ref. 5).

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2. Background leading up to the production of the antivenom for the redback spider.

The expansion of the habitat of the redback spider in Japan and the increase in cases of bites indicate an increase in opportunities for bites among a wide range of age groups. The public is concerned because severe cases at the time of the bite are accompanied by general symptoms and severe pain, and also because the exotic toxic organism has expanded its habitat into the immediate environment, and stings are very painful and severe cases have been reported. The fact that no deaths have been reported in Australia due to the bite of the barking spider since the development of CSL’s antitoxin also increases the need for antitoxin products for treatment. In response to this situation, the Health and Welfare Science Research and AMED Research Fund “Research on Quality Control of Antivenom and Treatment Using Antivenom”: 123 Toru Ichijima (St. Luke’s International University), launched in 2013, is conducting a clinical study on the importation of Culex pipiens antivenom and adverse reactions when using domestically produced Culex pipiens antivenom Verification and research was conducted on the development of an assurance system as a countermeasure, consideration of smooth transport of antitoxin in the event of a bite, and stockpile locations.

The plan was to purchase a certain amount of CSL’s antivenin as a countermeasure for bites of the spotted spider, and to provide it to physicians as a treatment for any cases of bites. In the course of dealing with each rare disease as a research group, personal importation of foreign antitoxin products has been significantly delayed, and in consideration of one of the objectives of the research group, which is to ensure the safety and security of the public against bites from the Dall’s porpoise, the research group decided to produce the Dall’s porpoise antitoxin in Japan in the same way that it produces the mountain lion antivenin. In 2015, the decision was made to implement the production of a domestically produced C. sapidus antitoxin for the C. sapidus spider. It was envisioned that once the domestically produced saddle spider antitoxin was completed, it would be available in a stable supply and would also be sufficient to respond to outbreaks.

3. Preparation for Production of domestic Cercopithecus antivenom

The Australian company CSL, which manufactures antivenin for horses, was unable to provide the information necessary for the domestic production of antivenin using horses, despite requests through the National Institute of Chemistry and Serum Therapy (KAKETSUKEN), a joint researcher. In 1961, a document published in the literature reported an increase in antibody titer in horses immunized with a toxin mixed with aluminum phosphate (ref. 6). We planned an immunization program and a quality control study, and first conducted a study of test immunizations with the saddleback spider antitoxin using rabbits. Specifically, three immunization methods were compared: 1) a combination of formalin-inactivated toxin and oil adjuvant, 2) a combination of untreated toxin and oil adjuvant, and 3) untreated toxin and Blood samples were collected from rabbits immunized with the three immunization methods, and the immunization methods were evaluated based on the titers of neutralizing antibodies against the saddle spider toxin.

First, we collected the saddleback spider crude toxin for the creation of immunizing antigens. A total of 11,524 female spiders (10,186 in Osaka and 1,217 in the suburbs of Nishinomiya City) were captured in cooperation with the Osaka Pest Control Association and the Nishinomiya City Environmental Health Division through the Department of Entomology and Medical Science of the Institute of Infectious Diseases, which is a joint researcher of the project. Ten hundred and seventy-seven females were dissected at the Faculty of Entomology and Medical Science, and toxins were extracted from the venom glands (Fig. 3), which were individually excised and purified to make one lot. Protein levels were quantified with some of the toxins and ranged from 1.5-2.5 mg/ml. The final lot was estimated to contain approximately 236 mg of protein. A total of 170 ml of toxin crude was obtained.

Formalin inactivation was performed to prepare the antigen for immunization, since a portion of the captured and purified toxin solution was used in rabbit immunization for preliminary studies. Two types of adjuvants for immunostimulation were studied: oil adjuvant and aluminum phosphate. Rabbits were immunized by each of the three immunization methods described above, and neutralizing antitoxin titers were measured in the sera of rabbits immunized against the red moss spider toxin. The highest antibody titer was obtained by immunization with the combination.

4. Titer test method of the antivenom of the redback spider and the bovine immunization method.

The Immunology Department of the National Institute of Infectious Diseases (NIID) conducted a study of titer testing methods as a way to confirm the efficacy of domestically produced antitoxins. The purpose of this study was to confirm the properties of crude venom extracted from Japanese spiders collected in Japan and to establish a titer test for antitoxin. Protein content of the spider toxin was determined, and the percentage and stability of α-latrotoxin, the main component of the toxin, were confirmed by the usual method. Various toxin doses were then administered to mice and observed alive or dead for 10 days, and the LD50 was calculated. Furthermore, 40 μg of toxin was mixed with various dilutions of antitoxin, allowed to stand for 1 hour, and then intravenously administered to mice and observed for 10 days to determine the titer of the antitoxin. The results showed that the main component of the toxin, α-latrotoxin, was well preserved in all lots and was stable for long-term storage at room temperature or 4°C. The LD50 of the toxin was calculated and averaged 9.16899 μg. The titer of the expired antitoxin was determined based on the established titer test, and both were found to maintain the indicated titer even after expiration. Thus, a titer test was established for the C. sessilemorpha antitoxin. It was also found that the toxin remained stable and toxic for a long period of time (ref. 7).

In addition, an immunization plan was developed at KAKETSUKEN using four horses for the production of the sargassumo antitoxin. Referring to the results of basic tests on rabbits and the actual production of antitoxin products at KAKETSUKEN, the following four immunization methods were planned to evaluate the adsorption performance of aluminum adjuvant on the spider toxin, after examining the amount of toxin adsorbed by aluminum hydroxide and aluminum phosphate at two different PH conditions The following is a summary of the results of the study. (1) Toxin immunization: The immunization method described in the literature was cited, and the number of additional immunizations was set to four, taking into account the basic test results at the National Institute of Infectious Diseases (NIID). Although aluminum phosphate was used as an adjuvant in the literature, aluminum hydroxide was used because it showed higher toxin adsorption performance than aluminum phosphate in a preliminary study. (2) Toxoid immunization (additional quantitative immunization): The number of basic immunizations was set to three quantitative toxoid immunizations and the immunizing dose to a fixed amount of 3 mg, referring to the conventional antitoxin production conditions at KAKETSUKEN. (3) Toxoid immunization (additional immunization with increased dose): Additional immunization with increased dose of toxoid was added. (4) Mixed immunization with toxoid and toxin: Incremental immunization with toxin was considered. We designed a four-condition equine immunization plan in which toxin or toxoid was mixed with aluminum adjuvant or oil adjuvant as an adjuvant (Table 1).

5. Prototype production of dried Cercopithecus antivenom

1) Capture of the red moss spider for horse immunization and crude purification of its toxin, 2) Investigation of a titer test method using the toxin, 3) Preliminary tests using rabbits, and 4) Immunization using horses for production of the antitoxin. Production was started using the equine antitoxin production facility at KAKETSUKEN with the support of AMED research funds, following the same procedure as the request for production by the research group in 2000. The immunoglobulin fraction was purified from horse plasma with relatively high titer of neutralizing antibody against the toxin, and the stock solution was obtained. After the dilution and addition of stabilizers, a prototype was lyophilized and manufactured. Reference specifications were established for these stock solutions and prototypes with reference to existing antitoxin products, and quality testing was conducted. The titer test was performed by a subcontracted researcher in the Immunology Department of the National Institute of Infectious Diseases (NIID). The reference standards for the test items other than the titer test were established based on the standards for existing snake venom antivenoms, and the tests were conducted in accordance with the Biological Preparation Standards or the Japanese Pharmacopoeia, respectively. Dried saddleback spider antivenin: Approximately 6,000 prototypes (titer 500 U/dose) were produced using a portion of the purified stock solution, and the unused horse plasma and purified stock solution were frozen and stored at -80°C. The purified stock solution was then used as a reference standard for the dried saddleback spider antivenin. The results of the quality test showed that the purified stock solution conformed to the reference standard established in the negative test for stray virus, and the prototype also conformed to the reference standard (Table 2: Ref. 8).

Neutralizing antibody titers were then measured for the produced sessile spider antitoxin. The neutralizing antibody titers by immunoassay are shown in Table 2. It was observed that the antibody titer increased after the basic equine immunization. Therefore, additional immunizations were conducted by several methods after the suspension period as scheduled. Among the immunization methods, the highest neutralizing antibody titer was obtained by increasing the amount of antigen in the additional immunization. It was also confirmed that sufficient titer was maintained in the undiluted solution and small lyophilized product through the formulation process. To evaluate the stability of the formulation in nonclinical studies, titers were measured after 6 hours of exposure at room temperature after dissolution, and no decrease in titer was observed even after 6 hours of exposure. titer was measured. It was also evident that plasma antibody titers rapidly declined when partial blood samples were taken after the immunization was completed. The results showed that the plasma titers of the antivenin were lower than 1/3 of those of the Yamagakashi antivenin produced in 2000, and that the titers were higher than those of the Yamagakashi antivenin produced in 2000. A high specific activity antitoxin formulation with low protein content and high activity was produced. We believe that the fact that we were able to produce such a high quality product using an antigen that has never been used before, the saddle spider toxin, and with very little information on its production is due to the technical capabilities of KAKETSUKEN, which has abundant experience in the production of horse antitoxin products.

6. Conduct GLP-compliant safety studies.

A GLP safety study was conducted on the manufactured sessile spider antitoxin. The research group discussed the need for at least a GLP-compliant non-clinical study and a study equivalent to a Phase I study to administer a domestically produced antitoxin, which has not been administered to humans, to patients for the first time. Although a non-GLP non-clinical safety study of domestic antitoxin was conducted in the manufacturing year and a certain level of safety was confirmed, it was decided to conduct a GLP study of domestic antitoxin after receiving support from the MHLW Tuberculosis and Infectious Diseases Division and AMED that information from the GLP study would be useful in consideration of future administration to healthy subjects and patients.

The GLP test was established as a standard for applying the test to correctly evaluate the safety in the development of new pharmaceutical products, based on the reflection of the past drug-related accidents. The basic structure of the GLP stipulates the establishment of a reliability assurance department, clarification of the responsibilities of the contractor when the testing is outsourced to an external facility, structural equipment of the testing facility, preparation of standard operating procedures, animal management, and preparation of protocols and final reports. Each facility capable of conducting such GLP studies is subject to periodic compliance inspections by PMDA.

For the Japanese silkmoth spider antitoxin, we commissioned a GLP testing facility, Shin Nihon Kagaku Co. First, the following six tests were planned as the specific items required for the GLP study: I. Titer test method review test; II. Characterization validation test: test including validation of assay methods such as protein concentration, purity measurement, and titer test; III. Characterization stability test: test to confirm the stability of appearance, pH, and pH, and to confirm the stability of the test method; IV. Protein concentration, purity, and potency studies; IV. Dosage solution stability studies: storage conditions and shelf life are secured; V. Local muscle irritation study in rabbits; and VI. Intermittent repeated intravenous administration study in rats.

1. As a review of the titer test method, a titer test method using mice was conducted as an analytical item for dried C. siceraria antitoxin in the planned GLP-compliant toxicity study of dried C. siceraria antitoxin, and the validity of the established titer test method was confirmed.
2. As a characterization test method validation study, the purity test method (SE-HPLC method), quantification method (BCA method), and titer test method were validated to conduct the characterization test of dried C. siceraria antitoxin. The HPLC and BCA methods with ultraviolet external detection were validated to determine the purity and concentration of dried C. siceraria antitoxin in the preparation.

III.Characteristic stability test：Characterization tests [items: properties (appearance), analogues and purity (SE-HPLC method), quantitation (BCA method), and potency] of dried C. siceraria antitoxin were conducted to confirm its properties and stability during storage as appearance, pH, protein concentration, purity test and potency test.

1. Dose solution stability studies were conducted to determine the stability of dried Cercopithecus antivenom in PBS-mediated preparations for 24 hours refrigerated + 2 hours at room temperature.

V. In a local muscle irritation study in rabbits, 1.0 mL/site of dried C. siceraria antitoxin was intramuscularly administered twice at 2-hour intervals to the lateral vastus muscle of the thigh of male Japanese White rabbits, and the local muscle irritation was examined at 2 and 14 days after administration. During the observation period, no deaths occurred in all patients, and there were no abnormalities in general condition or body weight trend. No erythema/crust and edema were observed at the site of administration in all patients in each group.

1. As an intermittent repeated intravenous administration study in rats, this toxicity study was conducted primarily to evaluate the risk of serum sickness with this drug, since antitoxin products carry a risk of serum sickness. The drug was administered into the tail vein of rats twice at 2-week intervals, and necropsy was performed at 2 and 28 days after the second dose for gross observation and evaluation. Histopathological observation of the autopsied rat whole body tissues was performed as an examination item. No deaths occurred in all cases during the observation period, and no toxic changes that could be attributed to the test substance were found in the general condition, body weight, food intake, ophthalmologic examination, urinalysis, hematologic examination, blood biochemical examination, necropsy, organ weights, and histopathologic examination. The results of all GLP-compliant safety tests from I to VI, which were reviewed in this way, showed that the Japanese-made saddle spider antitoxin produced in this study was free of any problems in any of the items (Reference 9).

7. Conclusion

In order to respond to severe cases of bites by the Japanese barking spider, an invasive alien species designated by the Ministry of the Environment and which has expanded its habitat to almost the entire country in the 25.6 years since it was first discovered in Japan, we have produced a domestically produced barking spider antitoxin and completed GLP-compliant safety tests without any problems. In order to use this domestically produced antitoxin in humans, another condition was to conduct the equivalent of a Phase I clinical trial, the results of which would have made this drug available for clinical research by the Antivenom Group, but no Phase I clinical trial has been conducted. The study plan was based on the consideration that this antitoxin is an unapproved drug and that a safety study of the drug is essential for its administration to humans. However, due to the expansion of the research content, including the change of representative after the three-year break of the research group, as well as the discussion on the positioning of this antitoxin to become an approved drug, no human administration study has been conducted.

Three years have passed since this antitoxin was manufactured. Unlike foreign antitoxin preparations, the expiration date of the lyophilized, domestically manufactured Huma antitoxin preparation is stable and stipulated to be 10 years. In addition, although the mountain lion antivenin, one of the objectives of the research group, has been in production for 20 years, no significant deterioration in its quality has occurred. The results of quality control tests of a Japanese-made saddle spider antitoxin formulation after three years of production also showed no deterioration in its quality. Although a large amount of domestically produced antitoxin, which is expected to be more potent than foreign products, is being manufactured, none is being used. It is hoped that these antitoxins will be made available to the public so that they will not have to rely on foreign products whose supply is unstable. To date, we have been able to import and stockpile some antivenin from overseas. Although the bites themselves have occurred, fortunately there have been no serious cases during this period for which the antitoxin should be administered therapeutically. However, the procurement of antitoxin products from overseas is not stable, and the supply is only available on a tightrope by looking for import partners each time the expiration date approaches.

Since the bite of the Drosophila melanogaster is a very rare disease, and clinical trials in humans are virtually impossible, close consultation between the responsible manufacturer, the PMDA, and the Ministry of Health, Labour and Welfare is required before this antitoxin preparation can be published as an approved drug, which is an ongoing issue for the study group.

Fig. 1: Female of the red-bellied spider

From the 2014 AMED report

Figure 2: Distribution of the redback spider in Japan

From the Ministry of the Environment website

Figure 3. venom gland of a redback spider (white tissue following a pair of brown fangs)

From the 2014 AMED report

Table 1. bovine immunization plan for antigens for the immunization of the western blotch spider.

No.1 Toxic immunization

No.2 Toxoid immunization (quantitative additional immunization)

No.3 Toxoid immunization (incremental immunization)

No.4 Toxoid and toxin mixed immunization

From the 2015 AMED report

Chart

Reference data

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