Minimum Lethal Dose Research Articles

Insecticidal and neuroblocking potencies of variants of the thiazolidine moiety of thiacloprid and quantitative relationship study for the key neonicotinoid pharmacophore

The pharmacophore of the neonicotinoid insecticide thiacloprid, cyanoiminothiazolidine, was modified to heterocycles such as imidazolidine, pyrrolidine and oxazolidine (the central ring hereafter). Their 6-chloro-3-pyridylmethyl or 5-chloro-3-thiazolylmethyl derivatives were examined for insecticidal activity against the American cockroach by injection and neuroblocking activity using the cockroach ganglion. The derivatives showed strong insecticidal activity with the minimum lethal dose (MLD) of about 10 nmol, which were however mostly weaker than the corresponding nitromethylene or nitroimine compounds. The activity was enhanced in the presence of synergists. The neuroblocking effect of cyanoimino compounds was at the micromolar level. Quantitative analysis for 23 variants of the key pharmacophore, constructed with the central ring conjugated to an NCN, CHNO2, or NNO2, showed that the neuroblocking potency is proportional to the Mulliken charge on the nitro oxygen atom or cyano nitrogen atom. The optimum log P value was evaluated as 1.19. The equation for the insecticidal- vs. the neuroblocking-potencies indicated that both potencies are related proportionally with each other when the other factors are the same.

Utilization of Bombyx mori larvae as a surrogate animal model for evaluation of the anti-infective potential of oxazolidinones

Silkworm (Bombyx mori) larvae were investigated as an alternative animal model for the efficacy testing of novel oxazolidinones. The minimal lethal dose (MLD) for Staphylococcus aureus was 1-5 x 10(7) CFU per larva, exhibiting more than 90% mortality within 2 to 4 days post-infection. Treatment with vancomycin, linezolid, and novel oxazolidinones (RBx 7644, RBx 8700, and RBx 2006171) showed survival in a dose-dependent manner. The antibacterial potential of RBx 7644 and RBx 8700 was compared with that of linezolid and that of vancomycin and the effective doses (ED)50 obtained in the silkworm model were 37.89, 24.96, 13.38, and 30.72 mg/kg body weight, respectively. The ED50 values showed a similar trend in a murine model of infection. Owing to the small size of the larvae, very small amounts of new chemical entities (NCEs) are required to test their in vivo efficacy in this model. Thus, the silkworm model may be used in the early stage of new discovery research.

Evaluation of the neutralizing capacity of Androctonus crassicauda (Olivier, 1807) antivenom against Leiurus quinquestriatus (Ehrenberg, 1928) venom (Scorpiones: Buthidae)

The two most venomous species of the family Buthidae, Leiurus quinquestriatus and Androctonus crassicauda, are found in Africa and in the Middle East. Potency and paraspecific activities of A. crassicauda antivenom (RSHC anti-Ac) were tested against L. quinquestriatus venom. The sera produced by Refik Saydam Hygiene Center (RSHC) showed strong reactivity against the venoms of A. crassicauda and L. quinquestriatus in western blotting and dot-blot analysis. RSHC anti-Ac presents immunoactivity and neutralizing potential against Leiurus quinquestriatus venom. Neutralization capacity of antivenom was found to be 400 µL against 40 minimum lethal doses (MLD) of A. crassicauda scorpion venom and 10 MLD of L. quinquestriatus venom. This study indicates that the RSHC anti-Ac could be used for treating L. quinquestriatus stings.

Pharmacophore of neonicotinoid insecticides

Our recent study related to neonicotinoid pharmacophore was presented. The first topic described on the QSAR study with 17 variants of 2-nitroiminoimizolidine. The insect neuroblocking potency is proportionally related both to the Mulliken charge on the nitro oxygen atom and log P. The second raised the functional possibility of the 3-fluoropropyl group as an H-bond acceptor for the neonicotinoid skeleton. Its 2-nitroiminoimidazolidine derivative actually showed the insecticidal tendency common to the neonicotinoids bearing a 6-chloro-3-pyridylmethyl group, although the activating strength was not as high as expected. The third introduced a new type of neonicotinoid compounds of alkylene-tethered twin-structure. The insecticidal activity against American cockroaches was dependent on the tether length and maximized at the hexamethylene with the minimum lethal dose of 2.2 nmol/insects, comparable to that of imidacloprid.

A European pharmaceutical company initiative challenging the regulatory requirement for acute toxicity studies in pharmaceutical drug development

Regulatory guidelines indicate acute toxicity studies in animals are considered necessary for pharmaceuticals intended for human use. This is the only study type where lethality is mentioned as an endpoint. The studies are carried out, usually in rodents, to support marketing of new drugs and to identify the minimum lethal dose. A European initiative including 18 companies has undertaken an evidence-based review of acute toxicity studies and assessed the value of the data generated. Preclinical and clinical information was shared on 74 compounds. The analysis indicated acute toxicity data was not used to (i) terminate drugs from development (ii) support dose selection for repeat dose studies in animals or (iii) to set doses in the first clinical trials in humans. The conclusion of the working group is that acute toxicity studies are not needed prior to first clinical trials in humans. Instead, information can be obtained from other studies, which are performed at more relevant doses for humans and are already an integral part of drug development. The conclusions have been discussed and agreed with representatives of regulatory bodies from the US, Japan and Europe.

A hand-held surface plasmon resonance biosensor for the detection of ricin and other biological agents

There is an ongoing need for field-deployable biosensor devices. We have constructed a fully self-contained, hand-held biosensor, based on the surface plasmon resonance technique. The dimensions of the sensor unit are 15 × 8 cm, the weight is 600 g and it is powered by a 9 V battery. We have characterised the responsiveness of the sensor using calibrated sucrose solutions and were able to measure changes as small as 3.3 × 10 −6 refractive index units. To demonstrate functionality of the sensor, we have prepared surfaces with an antibody fragment specific for the biological toxin ricin. We were able to detect ricin at 200 ng/mL in 10 min, which is approximately 2500 times less than the minimum lethal dose. We were also able to verify positive binding within a second 10 min window. This sensor demonstrates important steps required for the development of fully integrated, hand-held sensor devices and will form the basis of a multi-analyte system, to be developed in the near future. It also represents the first completely hand-held SPR device, not requiring external power or a computer connection to operate.

De novo-derived cationic antimicrobial peptide activity in a murine model of Pseudomonas aeruginosa bacteraemia

We describe the antimicrobial activity against Pseudomonas aeruginosa of the de novo-derived antimicrobial peptide WLBU2 in an animal model of infection. For this study, an intravenous (iv) model of P. aeruginosa infection was established. The minimum lethal murine dose of P. aeruginosa strain PA01 was determined to be 3 x 10(7) cfu when bacteria were administered iv. Increasing concentrations of WLBU2 were instilled either prior to or following PA01 septic exposure. For the mice given peptide post-bacterial infection, in the 1 mg/kg group, nine of nine animals died because of Pseudomonas sepsis; in the 3 mg/kg group, only one of nine succumbed to infection and in the 4 mg/kg group, all mice were protected (P < 0.0001). Similar results were obtained when WLBU2 was given 1 h prior to Pseudomonas infection. Although the therapeutic window in this model is narrow, the results nonetheless provide encouraging evidence for WLBU2 as a potential prophylactic or treatment of bacterial infection.

Proinsecticide candidates N-(5-methyl-2-oxo-1,3-dioxol-4-yl)methyl derivatives of imidacloprid and 1-chlorothiazolylmethyl-2-nitroimino-imidazolidine

Prodrugs of imidacloprid and the thiazolylmethyl analog masked with oxodioxolylmethyl group on the N3 site were prepared. The prodrugs decomposed in a buffer solution of pH 8.3 and in a physiological salt solution with half-lives of 10–15h, releasing the parent insecticides. Being consistent with this, an inward current was evoked in dissociated cockroach neurons treated with the masked compound solutions, which were maintained for 24h after preparation, as measured using patch–clamp electrophysiology, whereas no response was observed in neurons when the solutions were challenged immediately after preparation. The insecticidal test on the American cockroach showed that the minimum lethal dose for each compound at 24h after injection was 6.4×10−8mol, which was similar to that for imidacloprid and the thiazolyl derivative. This result strongly suggested a regeneration of the active ingredients in vivo.

Proteins, lethality and in vivo effects of Iurus dufoureius asiaticus scorpion venom

Iurus dufoureius asiaticus, Birula, 1903 scorpions were collected in Mugla province located in the Aegean region, Turkey. There are few number of publications about I.d. asiaticus, and there are no data regarding minimal lethal dose and effects of the scorpion venom till now. This is the first study about toxicity and effects of I.d. asiaticus scorpion venom in mice. Previously, most of the proteins in venom of I.d. asiaticus from Aydin region in Turkey were reported to be between 14 and 205 kDa in size. In this study, we determined the electrophoretic protein pattern of the venom taken from Mugla province to be between 29 and 116 kDa by SDS-PAGE. Intracerebroventricular (i.c.v.) was determined instead of s.c. injection since there were no deaths in any s.c. test groups. The LD 50 of I.d. asiaticus scorpion venom was found to be 47.7 μg/20 g mouse by i.c.v. injection route. After s.c. injection venom, mice were shown any intoxication symptoms. On the other hand, after i.c.v. administration of venom, mice showed symptoms such as excitability, hyper salivation, weakness, paralysis, coma and resulting in death. The possible cause of death could be due to multi-system organ failure depending on the toxic effect of the venom. These both results showed that the venom was not lethal on s.c. injection, but it was lethal on i.c.v. injection. This may imply that the scorpion is of little danger to humans.

Antiplasmodial Activity of Aryltetralone Lignans from Holostylis reniformis

Extracts from Holostylis reniformis were tested in vivo against Plasmodium berghei and in vitro against a chloroquine-resistant strain of Plasmodium falciparum. The hexane extract of the roots was the most active, causing 67% reduction of parasitemia in vivo. From this extract, six lignans, including a new (7'R,8S,8'S)-3',4'-methylenedioxy-4,5-dimethoxy-2,7'-cyclolignan-7-one, were isolated and tested in vitro against P. falciparum. The three most active lignans showed 50% inhibitor concentrations of < or =0.32 microM. An evaluation of minimum lethal dose (30%) values showed low toxicity for these lignans in a hepatic cell line (Hep G2A16). Therefore, these compounds are potential candidates for the development of antimalarial drugs.

C Terminal Half Fragment (50 kDa) of Heavy Chain Components of Clostridium botulinum Type C and D Neurotoxins Can Be Used as an Effective Vaccine

Recombinant whole heavy chains (H, 100 kDa) and their N-terminal (Hn, 50 kDa) and C-terminal (Hc, 50 kDa) half fragments of Clostridium botulinum type C and D neurotoxins were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. GST eliminated-preparations of H (10 microg), Hn (5 microg), Hc (5 microg), or a mixture of Hn (5 microg) and Hc (5 microg) of types C and D were mixed with an equal volume of adjuvant, and then were twice injected into mice subcutaneously. After immunization, the mice were challenged with up to 10(6) the minimum lethal doses (MLD)/0.5 ml of C or D toxin, the type of which was same as that of the immunogens. All of the mice immunized with antigens except for Hn survived against 10(5) to 10(6) MLD/0.5 ml of the toxins, but the mice immunized with Hn were killed by 100 MLD/0.5 ml. The mice immunized with a mixture of C-Hc and D-Hc, each 5 microg, also showed a high level of resistance against both C and D toxins. Antibody levels immunized with GST fused-or GST eliminatedpreparation were quite similar. These results indicate that recombinant GST-fused Hc can be used as a safe and effective vaccine for type C and D botulism in animals. It also became clear that one time inoculation with a large amount of C-Hc or D-Hc, 100 microg, is useful for vaccine trials in mice.

A novel toxin homologous to large clostridial cytotoxins found in culture supernatant of Clostridium perfringens type C

An unknown cytotoxin was identified in the culture supernatant of Clostridium perfringens type C. The cytotoxin, named TpeL, which was purified using mAb-based affinity chromatography, had a lethal activity of 62 minimum lethal dose (MLD) mg(-1) in mice and a cytotoxic activity of 6.2x10(5) cytotoxic units (CU) mg(-1) in Vero cells. The nucleotide sequence of TpeL was determined. The entire ORF had a length of 4953 bases, and the same nucleotide sequence was not recorded in the GenBank/EMBL/DDBJ databases. The molecular mass calculated from the deduced amino acid sequence was 191 kDa, and a signal peptide region was not found within the ORF. The deduced amino acid sequence exhibited 30-39 % homology to Clostridium difficile toxins A (TcdA) and B (TcdB), Clostridium sordellii lethal toxin (TcsL) and Clostridium novyi alpha-toxin (TcnA). The amino acid sequence of TpeL is shorter than these toxins, and the homologous region was located at the N-terminal site. Eighteen strains of C. perfringens types A, B and C were surveyed for the presence of the tpeL gene by PCR. The tpeL gene was detected in all type B (one strain) and C strains (five strains), but not in any type A strains (12 strains). TpeL was detected in culture filtrates of the five type C strains by dot-blot analysis, but not in the type B strain. It was concluded that TpeL is a novel toxin similar to the known large clostridial cytotoxins. Furthermore, the data indicated that TpeL is produced by many C. perfringens type C strains.

Difference in the level of paralytic shellfish poisoning toxin accumulation between the crabs Telmessus�acutidens and Charybdis�japonica collected in Onahama, Fukushima Prefecture

The difference in paralytic shellfish poisoning (PSP) toxin accumulation in the crabs Telmessus acutidens and Charybdis japonica was investigated at Onahama, Fukushima Prefecture, from 2002 to 2005. The level of toxin accumulation in the hepatopancreas of T. acutidens corresponded to that of mussels when examined on a yearly basis. In 2003, some crabs had a high toxicity of approximately 1000 MU, which compares to one-third of the human minimum lethal dose. Therefore T. acutidens should be monitored as a vector species of PSP toxin. The toxin profile of T. acutidens was also investigated. Because an increase in highly toxic species of PSP toxins with a decrease in low toxic species, such as N-sulfocarbamoyl-11-hydroxysulfate toxins, was not clearly observed between consecutive samples, toxin transformation in T. acutidens was considered to have a minimal impact on toxicity. PSP toxins were also detected in several specimens of C. japonica, but the highest toxicity was only 7.4 MU/g in the hepatopancreas. Because C. japonica is widely distributed in the coastal waters of Japan, investigation of toxin accumulation in the crab should be continued, including its investigation in different areas of water from Onahama, where the causative dinoflagellates of PSP occur in different conditions.

Paralytic shellfish poison as an attractant for toxic snails

The attracting effect of paralytic shell poison (PSP) on eight toxic snail species (Polinices didyma, Natica lineata, N. vitellus, Zeuxis sufflatus, Niotha clathrata, Oliva miniacea, O. mustelina, O. hirasei) and two non-toxic species (Pomacea canaliculata, Satsuma bairdi) was investigated. Eight toxic species were reported to contain tetrodotoxin, and three out of these contained minor PSP. The minimum lethal dose of PSP in most toxic gastropods was estimated to be more than 150 MU PSP/20 g body weight, but in non-toxic gastropods was less than 15 MU PSP/20 g body weight. After the attracting test, all toxic gastropods showed significant positive linear relationship (Y=3.716X+0.363, r=0.8427) between comparative attracting variation and the toxicity reported. The relationship between PSP resistance ability and the toxicity also had a positive correlation (Y=0.91X+210.090, r=0.6803). However, the non-toxic species showed a negative response. This indicates that PSP is an attractant for toxic snails.

Determination of potency and paraspecific effects of Androctonus crassicauda (Olivier, 1807) antivenom against Mesobuthus gibbosus (Brullé, 1832) venom (Scorpiones: Buthidae)

Scorpion envenomation remains a real health problem in many countries. In scorpionism cases, it is often recommended that patients be treated with species-specific antivenom. Androctonus crassicauda venom has been used as antigen for antivenom production in Turkey, where this antivenom, called Turkish antivenom, has also been effective in the treatment of envenomation caused by species other than A. crassicauda. The present study aimed at determining the paraspecific effects and potency of the Turkish antivenom against Mesobuthus gibbosus (Brulle, 1832) venom. To assess the venom toxicity and the antivenom efficacy, we determined the Minimum Lethal Dose (MLD) and the Minimum Effective Dose (MED) instead of LD50 and ED50, respectively. Androctonus crassicauda antivenom was capable of neutralizing M. gibbosus venom (20 MLD). This was the first study indicating that A. crassicauda antivenom can be used for the treatment of Mesobuthus gibbosus stings, especially in Aegean Region, Turkey.

Quantitative measurement of cyanide released from Prussian Blue

Background. Prussian Blue (PB), ferric hexacyanoferrate is indicated for (oral) treatment of internal contamination with radioisotopes of cesium or thallium. Cyanide is 35–40% of PB's molecular composition, thus cyanide may be released during transit through the digestive tract under physiological pH. The U.S. Food and Drug Administration investigated the issue of cyanide release prior to drug approval to ensure the drug's benefits exceeded risks. Objectives. To determine cyanide released from PB under pH conditions that bracket human physiological exposure. Methods. PB was incubated in situ at pH 1.0–12, 37°C for 1–48 hours. Cyanide was measured using a validated colorimetric method by UV-VIS spectroscopy. Results. PB had the highest cyanide release at pH 1 (135 ug/g) and lowest release at pH 5.0–7.0 from the highest daily dose of PB (17.5 g) (21 ug/g). Considering the minimal lethal dose of cyanide is approximately 50 mg, the maximal cyanide released (1.6 mg) does not present a safety concern.

Effect of substituents at the 5‐position of the pyridine ring of imidacloprid on insecticidal activity against Periplaneta americana

The insecticidal activities of imidacloprid derivatives with a wide range of substituents at the 5-position on the pyridine ring against American cockroaches, Periplaneta americana (L.), were measured by injection with and without synergists propyl 2-propynyl phenylphosphonate and piperonyl butoxide. The log(1/MLD) value (MLD = minimal lethal dose in mol) without synergists was 7.96 for the methyl derivative, and the values were lower for other derivatives. Synergists enhanced the potencies of all the compounds tested. Considering these compounds and those with other substituents at this position, the region for maximum activity was predicted to be in the conjunction of the pyridyl 6-chlorine atom with a lipophilic small group in the 5-position.

BcIV, a new paralyzing peptide obtained from the venom of the sea anemone Bunodosoma caissarum. A comparison with the Na + channel toxin BcIII

Sea anemones produce a wide variety of biologically active compounds, such as the proteinaceous neurotoxins and cytolysins. Herein we report a new peptide, purified to homogeneity from the neurotoxic fraction of B. caissarum venom, by using gel filtration followed by rp-HPLC, naming it as BcIV. BcIV is a 41 amino acid peptide (molecular mass of 4669 amu) possessing 6 cysteines covalently linked by three disulfide bonds. This toxin has 45 and 48% of identity when compared to APETx1 and APETx2 from Anthopleura elegantissima, respectively, and 42% of identity with Am-II and BDS-I and-II obtained from Antheopsis maculata and Anemonia sulcata, respectively. This neurotoxin presents only a weak-paralyzing action (minimal Lethal Dose close to 2000 μg/kg) in swimming crabs Callinectes danae. This appears to be a different effect to that caused by the type 1 sea anemone toxin BcIII that is lethal to the same animals at lower doses (LD 50 = 219 μg/kg). Circular dichroism spectra of BcIII and BcIV show a high content of β-strand secondary structure in both peptides, very similar to type 1 sodium channel toxins from various sea anemones, and to APETx1 and APETx2 from A. elegantissima, a HERG channel modulator and an ASIC3 inhibitor, respectively. Interestingly, BcIII and BcIV have similar effects on the action potential of the crab leg nerves, suggesting the same target in this tissue. As BcIII was previously reported as a Na + channel effector and BcIV is inactive over Na + currents of mammalian GH3 cells, we propose a species-specific action for this new molecule. A molecular model of BcIV was constructed using the structure of the APETx1 as template and putative key residues are discussed.

Detecting Clostridium botulinum

To the Editor: In the October 2005 issue of Emerging Infectious Diseases, Song et al. described a fiber-optic, microsphere-based, high-density array composed of 18 species-specific probe microsensors, used to identify biological warfare agents, including Clostridium botulinum (1). Although the researchers used multiple probes for C. botulinum, we doubt that this approach is suitable for this organism. C. botulinum comprises a heterogenous group of subspecies that produce botulinum neurotoxin (BoNT); identification and characterization usually rely on animal testing that focuses on antigenetically distinct toxins (2). Although strains of C. botulinum that do not produce toxins are sometimes isolated from wound infections not related to botulism, some strains of C. butyricum and C. baratii are also able to produce BoNTs. The mouse bioassay is currently the accepted method for detecting BoNT. In this assay, mice that receive an intraperitoneal injection containing a sample with more than a minimum lethal dose show symptoms of botulinum intoxication and die. ELISAs, which recognize protein antigenic sites, are still less sensitive than the mouse bioassay (3). Because the mouse bioassay requires euthanizing many animals, and results are not available for several hours, new diagnostic methods are needed. For C. botulinum, an organism widely dispersed in the environment, DNA-based methods may not provide the ultimate solution. Rapid methods to detect and differentiate active BoNTs, such as the rapid, mass spectrometry-based, functional method, are promising candidates to substitute for animal testing in the near future (4).

Immunomagnetic beads assay for the detection of botulinum neurotoxin types C and D

An immunomagnetic beads assay for the simultaneous quantification of botulinum neurotoxin types C and D was developed. Specific monoclonal antibodies against the heavy chain of the toxin and affinity-purified biotinylated polyclonal antibodies (pAbs) were used. The antibodies were preincubated with the sample. The complex being formed was then captured by magnetic beads coated with antimouse IgG. Streptavidin–poly-horseradish peroxidase, a signal amplifier, bound to the biotinylated pAb. A maximum sensitivity of approximately 0.3 minimal lethal doses for mice per milliliter was achieved with culture supernatants of both toxin types.