Preparation of high-drug-loading cantharidin polymer micelle delivery system and its anti-breast cancer activity / 中国药科大学学报

@#Abstract: The aim of this study was to prepare a high drug-carrying capacity micellar drug delivery system (CTD@Sol) of the polymer zebra tetracycline and to preliminarily investigate the feasibility of this drug delivery system for the treatment of breast cancer. Firstly, CTD@Sol was prepared using sol as the carrier material and CTD as the model drug, and its pharmacological properties such as appearance and morphology, particle size, potential and in vitro release were evaluated. The growth inhibitory and apoptotic effects of CTD@Sol on breast cancer (4T1) cells were investigated by MTT assay and Annexin V-FITC/PI double staining assay; the uptake efficiency of 4T1 to this delivery system was investigated by flow cytometry; and the in vivo tissue distribution of the delivery system and the targeting of tumour tissues were investigated by small animal in vivo imaging technique. The results showed that CTD@Sol appeared as a light pale blue creamy white colour, with an average particle size of (159.73 ± 1.96) nm, a PDI of 0.198 ± 0.006, Zeta potential of –(47.60 ± 1.77) mV, an encapsulation rate of (90.29 ± 1.69)% and a drug loading capacity of (45.00 ± 0. 84)%; the in vitro release and haemolysis experiments showed that the drug release rate of CTD@Sol in acidic environment (pH 5.5) was significantly faster than that in neutral environment (pH 7.4), suggesting that the system is acid-sensitive and has good biosafety under endocytosed pH conditions. Cellular uptake, cytotoxicity and apoptosis experiments showed that CTD@Sol was more lethal to 4T1 cells, and the sol-gel polymer micelles as a drug delivery vehicle could significantly improve the cellular uptake efficiency of the drug; in vivo experiments showed that the delivery system had a significant targeting effect on tumour tissues.In conclusion, this study has successfully produced a CTD@Sol drug delivery system with high drug loading capacity (>45%), good pharmacological performance, strong targeting and biosafety, which has the potential to be used in the treatment of breast cancer.

La cantarida, un antecesor del sildenafil / The cantarida, an antecesor of sildenafil

Resumen En todas las culturas y en todos los tiempos, brujos y alquimistas han buscado la fórmula que mejore, restablezca o potencie el vigor sexual. Mucho antes de la aparición del Sildenafil, existían preparados con una relativa eficacia, aunque en la mayoría de los casos, con un riesgo elevado que iba desde las lesiones severas hasta la muerte. Un preparado utilizado durante centurias en Europa que fuera introducido en la farmacopea americana fue el confeccionado a través de un insecto, la Lytta vesicatoria o cantárida. Se describen algunos de los usos históricos que se le dió a la cantaridina como afrodisíaco y los efectos que estos trajeron.

Abstract In all cultures and at all times, witches and alchemists have sought the formula that improves, restores or enhances sexual vigor. Long before the appearance of Sildenafil, there were preparations with relative effectiveness, although in most cases, with a high risk ranging from severe injuries to death. A preparation used for centuries in Europe that was introduced into the American pharmaco-peia was the one made from an insect, the Lytta vesicatoria or cantharide. Some of the historical uses of cantharidin as an aphrodisiac and the effects they brought are described.

Regulation of platelet function by cantharidin via PI3K/Akt/PKC pathway / 中国药理学通报

To investigate the effect of cantharidin ( CTD) on platelet function and the mechanism of anti-platelet aggregation. Methods Washed platelets were collected from the venous blood of healthy volunteers. The effect of CTD on platelet aggregation and release was determined by aggregometer. The CTD concentration was 2.5 ,5 ,10 μmol • L

Research Progress on Multiple Organ Damage and Mechanism of Cantharidin Poisoning / 法医学杂志

Cantharidin poisoning has been proven to cause multiple organ damage. Acute circulatory failure, acute renal failure, and multiple organ failure resulting from cantharidin poisoning are the main causes of death for patients with cantharidin poisoning. However, research on the damage of main target organs and mechanism of cantharidin poisoning is not clear. This paper reviews the latest toxicological and pathological research literatures at home and abroad related to cantharidin poisoning and comprehensively summarizes the latest research progress on the toxicological and pathological damage and mechanism of the digestive system, circulatory system, respiratory system, urinary system, reproductive system, skin mucosa, immune system, and nervous system after cantharidin poisoning, to provide reference for improving the molecular toxicological mechanism of cantharidin poisoning and decision-making in the clinical intervention of cantharidin poisoning.

Mechanism of Cantharidin-induced Morphological Changes and Dissociation of HCT116 Cells / 中国实验方剂学杂志

Objective:To explore the molecular mechanism of cantharidin(CTD) in inducing morphological changes and dissociation in human colon cancer HCT116 cells, in order to study the correlation with tumor metastasis and provide a new basis for clinical use of cantharidin. Method:Different concentrations of CTD (0, 2.5, 5, 10, 15, 20 μmol·L-1) were added to each hole to culture for 7 to 10 days, so as to determine the inhibitory effect of CTD on HCT116 cells; and changes of F-actin cytoskeleton and integrin in HCT116 cells were detected by immunofluorescence staining. Western blot analysis of protein expressions of RhoA,RhoB,RhoC,Cdc42 and Rac1/2/3 of Rho family were performed before and after cantharidin treatment. overexpression of RhoA was constructed by plasmid transient transfection, and effect of 10 μmol·L-1 cantharidin on the morphology and adhesion of overexpressing cells was also observed. Result:Cantharidin induced cytoskeletal remodeling and decreased integrin content in HCT116 colon cancer cells. RhoA protein was a member of Rho enzyme family with the greatest variation after cantharidin action. The proportion of transfected RhoA cells with green fluorescence was about 60%, the expression of RhoA protein in constructed RhoA overexpression cells was significantly increased, compared with wild-type HCT116 cells (PPConclusion:Cantharidin can inhibit the expression of RhoA protein, induce the morphological changes of HCT116 cells and weaken the adhesion to the basement, thereby inhibiting cell migration.

Optimization of prescription process of cantharidin nanostructured lipid carrier by central composite design-response surface methodology / 中草药

Objective: To integrate the toxic component of cantharidin (CTD) into a novel nanostructured lipid carrier (NLC) and optimize the cantharidin nanostructured lipid carrier (CTD-NLC) formulation process to reduce the toxicity of CTD and enhance its targeting. Methods: CTD-NLC was prepared by emulsified ultrasonic dispersion method. The encapsulation efficiency was determined by dialysis method. The average particle size, particle size distribution (polydispersity index, PDI), Zeta potential, encapsulation efficiency, and drug loading were taken as indicators. Univariate investigation and central composite design-response surface methodology (CCD-RSM) were used to optimize the prescription process of CTD-NLC. Multivariate quadratic fitting was used to evaluate the model equation between indicators and factors. The fitted equation was analyzed by the variance analysis and the optimal prescription was predicted by the resonse surface. Results: The optimized CTD-NLC prescriptions were as follow: mass of total lipid was 453.66 mg, solid to liquid lipid ratio of 1:2, total stable dose of 16.9 mg/mL, ratio of Pluronic F68 to egg yolk lecithin (Lipoid E PC S) of 3.88:1, with ultrasound for 30 min (working 2 s, stopping 2 s). The prepared CTD-NLC was clear clarification in appearance with light blue opalescence, the average particle size was (85.99 ± 0.49) nm, PDI was 0.280 ± 0.002, Zeta potential was (-8.21 ± 0.24) mV, encapsulation efficiency was (98.57 ± 0.05)%, and drug loading was (0.65 ± 0.01)%. Conclusion: The fitting model established by CCD-RSM is accurate and reliable. The optimized CTD-NLC distribution is concentrated, with high encapsulation efficiency and good physical stability. It lays a foundation for the subsequent in vitro and in vivo studies of CTD-NLC.

Research progress on derivative cantharidin bioactivities and toxic regulations / 中草药

Cantharidin is the main active component of traditional Chinese medicine Mylabris. Cantharidins received increasing attentions for having bioactivities such as anticancer, anti-inflammatory and enhancing leukocytes, meanwhile, many researches were reported about cantharidin bioactivities, structure modifications, and synthesis methods. In this review, structures of derivative cantharidins were summarized and bioactivity and toxic regulations were developed, which can provide reference for cantharidin compound researches and modernizations of Mylabris.

Preparation process of galactosylated liposome of modified cantharidin / 中草药

Objective To optimize the formulation ratio and preparation process of galactosylated cantharidin liposome (Lac-CTD- lips) and establish its methodology for content determination. Methods The method of determination of GC-MS cantharidin content was established by film dispersion method. The entrapment efficiency of cantharidin was evaluated as an index. The preparation process of Lac-CTD-lips was optimized by single factor and orthogonal experiments. Its surface characteristics, encapsulation efficiency, particle size, and Zeta potential were also investigated. Results The best prescription was as follow: cantharidin: hydrogenated soya lecithin:cholesterol at 1:20:5, 10% galactoside, film-forming at 50 ℃, film cleaning with 30 mL of PBS solution of pH 6.0, and hydartion at 40 ℃ for 1.5 h. The resulting liposomes exhibited a pale blue opalescent appearance, a spherical particle morphology, and a more rounded surface with no adhesion. The average particle size was (123.9 ± 4.8) nm (n = 3), the particle size distribution was single-peak, the zeta potential was (-0.36 ± 0.81) mV (n = 3), and the encapsulation efficiency was over 75%. Conclusion GC-MS is suitable for the determination and analysis of cantharidin content. The optimal preparation technology from orthogonal experiment is stable and reliable. The obtained liposomes have higher encapsulation efficiency, small particle size, and good appearance.

Content Determination of Cantharidin in Lytta caraganae by HPLC / 中国药房

OBJECTIVE:To establish the method for content determination of cantharidin in Lytta caraganae,and to use the result as the extract screening evidence of L. caraganae source material. METHODS:Ultrasonic extraction method was used to extract cantharidin from L. caraganae using acetone as solvent. HPLC method was adopted to determine the content of cantharidin. The determination was performed on C18column with mobile phase consisted of methanol-water(23:77)at flow rate of 1.0 mL/min. The detection wavelength was set 230 nm,the column temperature was set at 35 ℃,and sample size was 10 μL. The content of cantharidin in L. caraganae was determined and compared with the results of content determination by the method stated in Chinese Pharmacopeia(using chloroform as extraction solvent). RESULTS:The liner range of cantharidin were 0.2-1.0 mg/mL (r=0.998 8)with average methodology recovery rates of 101.1%(RSD=1.7%,n=6). The average content of cantharidin in L. caraganae was 0.932%(n=3),while the content of cantharidin was 0.793%(n=3)determined by the method stated in Chinese Pharmacopeia. Both were higher than the requirement of Chinese Pharmacopeia that the content of cantharidin in scource material of cantharidin was higher than 0.35%. CONCLUSIONS:Established method is accurate and reliable for the content determination cantharidin in L. caraganae. The content of cantharidin is up to the standard of Chinese Pharmacopeia,and can be used as source material for exacting cantharidin.

Formula and Preparation of Cantharidin Chitosan Bioadhesive Microspheres by Spray Drying / 中国药师

Objective: To optimize the formula and preparation technology of cantharidin chitosan bioadhesive microspheres.Methods: The microspheres were prepared by a spray drying method.A single-factor experiment was used to study the effect of chitosan with different molecular weight on the gastric mucosa adhesion rate were also studied, and the effects of different drug loading ratio, concentration of chitosan acetic acid solution and speed of peristaltic pump on the loading rate were also studied.The cantharidin entrapment efficiency as the studying index, a response surface method was used to further optimize the formula.Results: The best formula and preparation process of cantharidin chitosan bioadhesive microspheres were as follows: the concentration ratio of antharidin to chitosan was 19.83% , the concentration of chitosan acetic acid solution was 0.77% , and the peristaltic pump flow rate was 9.225 ml·min-1.With the best formula, the entrapment efficiency of cantharidin chitosan bioadhesive microspheres was 90.14%.Conclusion: The microsphere preparation by the spray drying method is stable and repeatable.Cantharidin chitosan bioadhesive microspheres have high entrapment efficiency and promising biological adhesion.

Cantharidin suppressed breast cancer MDA-MB-231 cell growth and migration by inhibiting MAPK signaling pathway

As an active constituent of the beetle Mylabris used in traditional Chinese medicine, cantharidin is a potent and selective inhibitor of protein phosphatase 2A (PP2A) that plays a crucial role in cell cycle progression, apoptosis, and cell fate. The role and possible mechanisms exerted by cantharidin in cell growth and metastasis of breast cancer were investigated in this study. Cantharidin was found to inhibit cell viability and clonogenic potential in a time- and dose-dependent manner. Cell cycle analysis revealed that cell percentage in G2/M phase decreased, whereas cells in S and G1 phases progressively accumulated with the increasing doses of cantharidin treatment. In a xenograft model of breast cancer, cantharidin inhibited tumor growth in a dose-dependent manner. Moreover, high doses of cantharidin treatment inhibited cell migration in wound and healing assay and downregulated protein levels of major matrix metalloproteinases (MMP)-2 and MMP-9. MDA-MB-231 cell migration and invasion were dose-dependently inhibited by cantharidin treatment. Interestingly, the members of the mitogen-activated protein kinase (MAPK) signaling family were less phosphorylated as the cantharidin dose increased. Cantharidin was hypothesized to exert its anticancer effect through the MAPK signaling pathway. The data of this study also highlighted the possibility of using PP2A as a therapeutic target for breast cancer treatment.

Stability of cantharidin and cantharidic acid in Mylabris aqueous solution / 中国中药杂志

To investigate the stability and the conversion rules of cantharidin and cantharidic acid contained in the Mylabris aqueous solution under different conditions. The contents of cantharidin and cantharidic acid under different conditions (pH, temperature and light) were determined by HPLC-TQ-MS. The results showed that the content of cantharidin was gradually decreased with the rising of pH value, while on the contrary, the content of cantharidic acid was increased gradually; after Mylabris aqueous solution with different pH values were placed at 25, 40 ℃ and 25 ℃ respectively for lighting for 90 days, the samples were collected for analysis. The results showed the contents of cantharidin and cantharidic acid were changed greatly in the first 10 days, mainly including the decrease of cantharidic acid and increase of cantharidin when the solution was acidic, and the increase of cantharidic acid and decrease of cantharidin when the solution was alkaline. After that, the contents of the above two components basically remained stable. This study revealed that pH was the main factor to affect the contents of cantharidin and cantharidic acid, and they could be converted into each other with the changes of pH value. High temperature and light can accelerate the speed of achieving such balance. The study can provide certain reference for the quality control of the medicines using the Mylabris as raw material.

Clinical Observation of Rh-endostatin Combined with Cantharidin Sodium Vitamin B6 in the Treatment of Advanced Non-small Cell Lung Cancer / 中国药房

OBJECTIVE:To investigate the clinical efficacy and safety of rh-endostatin combined with cantharidin sodium vita-min B6 in the treatment of advanced non-small cell lung cancer (NSCLC). METHODS:180 patients diagnosed as advanced NSCLC were divided into group A,group B and group C ,with 60 cases in each group according to random number table meth-od. 3 groups were all given gemcitabine+cisplatin(GP)chemotherapy plan;Group B additionally received Rh-endostatin injection 7.5 mg/m2 intravenously for 3 h,d1-14;group C was additionally given Cantharidin sodium vitamin B6 injection 40 ml intrave-nously,qd,d1-14,on the basis of group B. every 21 days for a cycle,evaluation of therapeutic effect after 2 cycles. The clinical benefit rate,quality improvement rate of life,time to progression (TTP) and the occurrence of ADR were observed in 3 groups. RESULTS:The clinical benefit rate of groups A,B and C were 40.0%,58.3%,71.6% respectively,the quality improvement rate of life in 3 groups were 28.3%,41.7%,56.7% respectively,the differences were statistically significant among those groups(P0.05). The rates of leukopenia,thrombocytopenia,nausea and vomiting in group C were significantly lower than those of group A and B,with statistical significance(P0.05). CONCLU-SIONS:Rh-endostatin combined with cantharidin sodium vitamin B6 can significantly improve the effectiveness of chemotherapy in patients with advanced NSCLC on the basis of the GP chemotherapy,while reduce the toxicity of chemotherapy drugs,improve the quality of life and prolong the survival time.

Impact of cantharidin sodium vitamin B6 on serum tumor necrosis factor-α and interleukin-12 in non-small cell lung cancer patients / 中国医师杂志

Objective To investigate the relationship between level of serum tumor necrosis factorα (TNF-α) and interleukin-12 (IL-12) and the antitumor effects of cantharidin sodium vitamin B6.Methods Forty advanced non-small cell lung cancer patients aged 30 to 80 were divided into experimental groups [paclitaxel + cisplatin (TP program) and sodium cantharidate vitamin B6] and control group (only TP program).Peripheral blood was drawn in the day of admission and the seventh day,the level of serum TNF-α and IL-12 were detected by method of enzyme-linked immunosorbent assay (ELISA),the results were analyzed.The World Health Organization (WHO) anticancer drugs in acute and subacute toxicity grading criteria were used to grade the chemotherapy side effects.Results The level of serum TNF-α and IL-12 were no significant difference on admission.After chemotherapy,serum TNF-α and IL-12 of experimental group was significantly higher than control group (P ＜ 0.000 and P ＜ 0.037,respectively).The serum TNF-α and IL-12 of experimental group had upward trend after chemotherapy.After 3 months of treatment,the remission rate was 85.7％ in experimental group,significantly higher than 57.8％ in the control group (P ＜ 0.05).White blood cells,and platelets decrease in the experimental group were significantly reduced than the control group (P ＜ 0.001 and P ＜ 0.009,respectively),and there are no significant difference in nausea and vomiting reaction between experimental and control groups.Conclusions The antitumor effects of cantharidin sodium vitamin B6 might be related to the high level of serum TNF-α and IL-12,cantharidin sodium vitamin B6 in combination with chemotherapy in patients can reduce white blood cell and platelet decline after chemotherapy.

Identification of Chemical Component Cantharidin from Chinese Blister Beetle by LC-MS/MS / 世界科学技术-中医药现代化

This study was aimed to verify the chemical component cantharidin from Chinese blister beetle by LC-MS/MS. Components were first processed by HPLC to choose the appropriate separation conditions before LC/MS/MS analysis. Through the positive and negative ions pre-scan, positive ions scanning way was selected in the scanning of each separate component. The scanned maps were obtained and the relative molecular mass was deduced. The results showed that 8 types of cantharidin compounds were identified, including three isomers. It was concluded that LC-MS/MS was able to determine the chemical component cantharidin from Chinese blister beetle rapidly and accurately, which was existed in the body by the form of combined amino acid.

Basic Knowledge and Research Progress on Cantharidin-based Molecules / 世界科学技术-中医药现代化

This study was aimed to summarize main-bone structure of cantharidin-based small molecules and facts about three typical candidates including its origin, physical-chemical properties and general synthetic approaches. Basic chemical and pharmacological information as well as development of anti-cancer activities, which were related to cantharidin, norcantharidin and their analogues were reviewed, especially the relationship between the structures and their inhibitory activity and selectivity of serine-threonine protein phosphatases PP1, PP2A and PP2B in cancer treatment. The designs and developments of new biological cantharidin-based small molecules were also reviewed.

Physicochemical Properties and Gastric Mucosa Irritation of Cantharidin-hydroxypropyl-β-cyclodextrin Inclusion Complex / 中草药·英文版

Objective To increase the solubility and relieve the mucous irritation of cantharidin (CA) by preparing cantharidin-hydroxypropyl-β-cyclodextrin (CA/HP-β-CD) inclusion complex.Methods The inclusion complex was prepared by co-evaporation method and characterized by differential scanning calorimetry (DSC),X-ray diffractometry (XRD),and nuclear magnetic resonance (NMR).Results The disappearance of CA as well as the shift of exothermic peaks shown in DSC results indicated the complexation phenomenon.XRD results showed that the crystalline CA pattern had disappeared,and in NMR results,H-5 shifted from δ 3.731to 3.695 after complexation and H-2 shifted from δ 3.626 to 3.598,which suggested that part of the drug had entered the HP-β-CD cavity to form an inclusion complex.The solubility increased 10.3 times after complexation and the mucous irritation of CA was relieved remarkably.Conclusion Through complexation with HP-β-CD,the solubility and dissolution rate of CA are improved significantly,and the irritation of musous is relieved.

The effects of protein phosphatase 2A inhibitors on the viability of pancreatic cancer cell and its mechanism / 中华消化杂志

ObjectiveTo investigate the effects of protein phosphatase 2A (PP2A) inhibitors on the viability of pancreatic cancer cell line PANC-1 and its mechanism.MethodsPANC-1 cells were treated with PP2A inhibitors Cantharidin or Okadiac acid.The activity degree of NF-κB pathway was tested by Western blot.NF-κB pathway was blocked from all sectors by PP2Acα plamid transfection,NF-κB inhibition of protein kinase α (IKKα) and NF-κB inhibitor α (IκBα) dominant negative mutant and p65 interfering plasmid.Cell viability was determined by MTT.ResultsPP2A inhibitors could induce phosphorylation of IKKα,further phosphorylation of IκBα and degradation and followed by the release of p65 into nucleus.When PP2Acα,IKKα dominant negative mutant and IκBα dominant negative mutant were overexpressed,or p65 was interfered,the inhibition rate of Cantharidin on cell viability decreased (31.85±13.37) ％,(23.48±8.98)％,(22.63±5.81)％ and (20.88±3.24)％respectively,and the inhibition rate of Okadiac acid on cell viability decreased (40.17 ± 11.65)％,(27.34±14.28)％,(24.85±3.39)％ and (27.08±3.81)％ respectively.ConclusionsPP2Ainhibitors play a role in preventing pancreatic cancer through PP2Acα/IKKα/IκBα/p65 pathway.

Influences of 4-amino-2-methy cantharidinmide on epileptiform electroencephalogram, GABA and GABAB receptor in convulsive rats / 中国药学杂志

OBJECTIVE: To investigate the anticonvulsion effect of 4-amino-2-methyl cantharidinmide (AMC) and its influences on epileptiform electroencephalogram (EcoG), contents of γ-aminobutyric acid (GABA) and GABAB receptor. METHODS: Rat model of penicillin-induced-convulsion (PIC) was established by intracortical (ic) penicillin (PNC) injection in rat motor cortex. Valproate (VPA) was used as the positive control drug. Convulsion seizure latency and racine behavior study graduations were used as indexes to evaluate the efficacy. RM6240C multi-channel biological signal collection-processing system synchronously recorded EcoG of convulsive rats after intragastric (ig) administration of AMC (25.0, 100.0 mg · kg-1). The effects on convulsion and epileptiform discharge were analyzed. The contents of GABA and the expression of GABAB receptor in the cortex and hippocampus regions of rats were determined by immunohistochemistry technique. RESULTS: AMC at the two doses and VPA could reduce epileptiform activities and discharge and prolong the latencies of epilepsy seizure, compared with the PIC group (P 0.05); compared with model control, the expression quantity of hippocampal GABAB receptor protein was significantly increased (P 0.05). GABA expression quantity in groups of larger dosage AMC and VPA were respectively higher than that in the normal group and the model group, and the GABAB receptor protein expression quantity also significantly increased (P < 0.05). CONCLUSION AMC can antagonize the convulsion seizure and inhibit the epileptiform discharge induced by penicillin in rats. The anti-convulsion mechanism of AMC is possibly related with increasing GABA content and GABAB receptor expression in cortex and the hippocampus. Copyright 2012 by the Chinese Pharmaceutical Association.

Origin, transfer and distribution of cantharidin-related compounds in the blister beetle Hycleus scabiosae

Cantharidin provides chemical protection for the coleopteran families Meloidae and Oedemeridae. In the present study, it was observed that cantharidin concentration in Hycleus scabiosae was slightly decreased from mated females (mean = 0.011 mg/mg of dry weight) to males (mean = 0.010 mg/mg) and considerably diminished in relation to virgin females (mean = 0.005 mg/mg). Significant concentrations of palasonin (21.69 ng/mg among virgins and 17.49 ng/mg in mated females) and palasoninimide (14.62 ng/mg in virgins and 9.17 ng/mg in mated females) were found in H. scabiosae. Palasonin, palasoninimide and cantharidinimide content of eggs were measured as 5.61, 7.69 and 7.80 ng/mg respectively. Surprisingly, males showed no trace of cantharidin-related compounds (CRCs); therefore CRCs in H. scabiosae could not be transferred from males to females and based on experiments employing its deuterated form, cantharidin is probably independently synthesized in females from the male nuptial transfer. An inseminated female incorporates about 38.5 ng of cantharidin (0.34 percent of the maternal content), 196.35 ng of palasonin (91.82 percent of maternal content) and 269.15 ng of palasoninimide (96.70 percent maternal content) into each egg mass during oviposition. It seems that eggs of this meloid species exploit a different array of protective chemicals by increasing the ratio of CRCs versus cantharidin. CRCs are less toxic than cantharidin; therefore, such compounds might have been deposited in eggs as a safer substitute for cantharidin to provide effective protection, but does not simultaneously harm the susceptible embryo.(AU)