Nonclinical safety evaluation of Insulin NPH, a biosimilar of Humulin NPH / 药物评价研究

Objective To investigate the toxic reaction,toxic organs or target tissues of protamine recombinant human insulin (Insulin NPH),and provide basis for clinical trials by single dose toxicity test in mice,repeated toxicity and immunogenicity of Beagle's dogs,and systemic active allergy in guinea pig.Methods ① Using maximum dose method,mice in single dose toxicity test were sc injected with normal saline (NS),vehicle,and Insulin NPH (2092-2488 IU/kg),the toxic reactions after injection were monitored.② In repeated toxicity study,Beagle's dogs were sc administrated with vehicle,the original (Humulin NPH,1.5 IU/kg)and different doses of Insulin NPH (0.5,1.0 and 1.5 IU/kg) for 30 d continuously,followed by a 14-d recovery.During the administration and recovery period,general observation,local irritation,body weight,anus temperature,blood glucose,and electrocardiogram (ECG) were checked,moreover,hematology,serum biochemistry and urine were detected.Also,organic weights and histopathological examination were conducted.Binding antibodies in dog serum were measured by indirect ELISA method in immunogenicity test.③ In systemic active allergy study,cavies were sc injected with low-and high-dose (4 and 12 IU/kg) Insulin NPH,normal saline and vehicle.Besides,ova as positive control was also included.After five times of sensitization test with above doses,the excitation reactions of iv injection with tripled sensitizing doses were observed.Results No obvious toxicity was observed in mice after injected with 165 times of usual clinical dose of Insulin NPH.Repeated toxicity study of Beagle's dogs revealed that 1.0 IU/kg was the no-toxic-effect dose (NOAEL) for Insulin NPH,which was equivalent to 2 times of clinical dose.No bindingantibodies were found in immunogenicity test.There was no obvious allergic symptom in the active systemic allergy study of guinea pig.Conclusion Under the experimental conditions,no serious toxicity of Insulin NPH is found.

Study on allergy of Astragaloside injection / 药物评价研究

Objective To evaluate the hypersusceptibility of Astragaloside injection on animal,and provide reference for clinical use with active systemic anaphylaxis (ASA),passive cutaneous anaphylaxis (PCA) and determination of serum sample titer.Methods ASA:Guinea pigs was ip with 0.4,1.6 mg/kg Astragaloside injection five times every other day.On the eleventh day after the last administration,the test substance was quickly injected to fore limb vein,and animal allergy symptoms were observed within 30 min.PCA:Astragaloside injection was ip injected to rats five times every other day and antiserum was collected.The antiserum was appropriately diluted,and sc injected to another group rats for passive sensitization.About 48 hours later,Astragaloside was quickly iv to rats,and the skin allergy was observed.Meanwhile,the antibody titer of the antiserum was determined.Results ASA:Astragaloside injection of 0.4,1.6 mg/kg in guinea pigs did not show any allergic reaction,that is,ASA was negative;PCA:Astragaloside injection of 0.5,2.0 mg/kg in rats did not show any allergic reaction,and Astragaloside specific antibodies were not determined in serum samples.That is,PCA was negative.Conclusion The results of ASA and PCA were negative in the experimental dose,and there was no specific antibody against Astragaloside in the serum prepared by PCA,which indicated that the possibility of hypersensitivity reaction was weak in clinical use.

Study on allergy of Astragaloside injection / 药物评价研究

Objective To evaluate the hypersusceptibility of Astragaloside injection on animal,and provide reference for clinical use with active systemic anaphylaxis (ASA),passive cutaneous anaphylaxis (PCA) and determination of serum sample titer.Methods ASA:Guinea pigs was ip with 0.4,1.6 mg/kg Astragaloside injection five times every other day.On the eleventh day after the last administration,the test substance was quickly injected to fore limb vein,and animal allergy symptoms were observed within 30 min.PCA:Astragaloside injection was ip injected to rats five times every other day and antiserum was collected.The antiserum was appropriately diluted,and sc injected to another group rats for passive sensitization.About 48 hours later,Astragaloside was quickly iv to rats,and the skin allergy was observed.Meanwhile,the antibody titer of the antiserum was determined.Results ASA:Astragaloside injection of 0.4,1.6 mg/kg in guinea pigs did not show any allergic reaction,that is,ASA was negative;PCA:Astragaloside injection of 0.5,2.0 mg/kg in rats did not show any allergic reaction,and Astragaloside specific antibodies were not determined in serum samples.That is,PCA was negative.Conclusion The results of ASA and PCA were negative in the experimental dose,and there was no specific antibody against Astragaloside in the serum prepared by PCA,which indicated that the possibility of hypersensitivity reaction was weak in clinical use.

Exploration on the selection of positive materials in active systemic anaphylaxis in guinea pigs / 中国比较医学杂志

ObjectiveTheaimofthisstudywastoprovideabetterpositivecontrolforallergictestbycomparing the allergic effect of two kinds of positive materials , human albumin and ovalbumin , on active systemic anaphylaxis in guinea pig.Methods Guinea pigs were randomly divided into 14 groups, and were given human albumin , ovalbumin (2, 10, 100 mg/animal), or 0.9%sodium chloride injection as test substances , to assess the symptoms and incidence of systemic allergic responses induced by different sensitizing substances in different challenge doses and different challenge intervals.Results In the range of 2 to 100 mg/animal, the guinea pigs showed a 100%incidence rate of positive allergic reaction to human albumin and ovalbumin , the severity of anaphylactic symptoms was increasing along with the increase of sensitizing doses and challenge doses , and the allergic reaction was more strong induced by the same dose of ovalbumin than human albumin .Conclusions Our findings indicate that in the active systemic anaphylaxis test in guinea pigs , we recommend ovalbumin as the positive control in a dose of 2 mg/animal.

Immunotoxicity study of Ginenoside Compound K Injection / 国际中医中药杂志

Objective Evaluate the immunotoxicity of Ginenoside Compound K Injection.Methods Active Systemic Anaphylaxis (ASA)tests and Passive Cutaneous Anaphylaxis (PCA)tests were used to evaluate Ginenoside Compound K Injection.Results In the ASA tests,positive control group showed pole-strength anapbylaxis,both the high-dose group and the low-dose group of Ginenoside Compound K Injection didn't produce allergic reaction and the body weights of all groups showed no significant differences.In the PCA tests,all rats of positive control group caused blue spots with their diameters bigger than 5 mm (diameters on the left side of blue spots was (10.1± 3.34) mm and diameters on the right side of blue spots was(7.57± 1.94)mm.Serum IgE was significantly increased.While both high dose and low dose of Ginenoside Compound K Injection group didn't show blue spots with their diameters greater than 5 mm and their IgE levels showed no significant differences compared with negative control group.Conclusion Ginenoside Compound K Injection showed no immunotoxicity under these experimental conditions.

Oral Tolerance in Active Fatal Anaphylaxis / 대한면역학회지

We have investigated whether oral administration of ovalbumin (OVA) could prevent active systemic anaphylaxis to the antigen. Oral tolerance was induced by a single feecfing with 40 mg OVA before, but not after, sensitization characterized by diminished OVA-specific IgE and IgG responses. Feeding 15 mg OVA suppressed anaphylaxis and antibody responses to a lesser extent. Spleen cells from tolerant donors were incapable of transferring the tolerance to naive recipients. Pretreatment of cyclophosphamide (100 mg/kg) 2 days before OVA feeding did not restore the tolerance. Furthermore, in vitro cell mixing studies showed that the proliferation of spleen cells from OVA- sensitized donors was not inhibited by the addition of spleen cells from tolerant donors, arguing against the role of suppressor cells. Anergy was demonstrated by the ability to reverse the tolerant state after culturing tolerant cells in the presence of IL-2. These findings indicate that only a high-dose (40 mg) feeding OVA was found to be effective in inducing tolerance in this experimental system, and demonstrate anergy as the mechanism underlying oral tolerance to systemic anaphylaxis.

Effects of Sensory Denervation by Neonatal Capsaicin Treatment on Cytokine Production and Various Immune Responses / 대한면역학회지

Capsaicin, the pungent principle of hot peppers, is a neurotoxin that depletes unmyelinated primary sensory neurons (polymodal nociceptors) of neuropeptides like tachykinins. However, the role of capsaicin-sensitive sensory nerve in the production of cytokines, penicillin V (PEV)-induced active fatal anaphylaxis and other immune responses is not yet fully established. Neonatal mice were pretreated s.c. with a single injection of 10 ug of capsaicin per mouse in volume of 20 ul within 5 days of age. Using 5-8 week old mice pretreated as neonates with capsaicin, the capsaicin- pretreated and vehicle-treated control mice were examined for various parameters of immune responses described above. For the induction of active fatal anaphylaxis with PEV, 8 week old mice pretreated as neonates and age-matched capsaicin- untreated control mice were sensitized i.p. with 500 ug of PEV-ovalbumin conjugate plus 2*10(9) B. pertussis and 1.0 mg alum and challenged i.v. with PEV-bovine serum albumin conjugate 14 days later. It was found that neonatal capsaicin-pretreatment significantly enhanced contact hypersensitivity to TNCB and hemagglutination response to SRBC, but significantly inhibited the proliferation response of rnurine splenocyte to Con A and LPS. Interestingly, neonatal capsaicin pretreatment significantly inhibited the intensity of PEV-induced active fatal anaphylaxis and decreased the mortality due to anaphylactic shock. It also significantly inhibited LPS- induced production of cytokines such as TNF-a, IL-1B, IL-6, IL-10, and IL-12. The capsaicin-pretreatment also resulted in an inhibition of the activation of NF-kB. Taken together, these data showed for the first time that neonatal capsaicin-pretreatment significantly inhibited an antibiotic (PEV)-induced anaphylaxis and production of various cytokines, and suggest that capsaicin-sensitive primary sensory nerve may play an important regulatory role in active fatal anaphylaxis and cytokine production, thus potentially presenting tools for immune intervention. In particular, the data presented also indicated the possibility to selectively down-modulate cytokine production and NF-kB activation may offer a broad application for therapeutic intervention in neuroimmunological diseases and other pathological situations.

Effect of Human Seminal Plasma on Cytokine Prodection and Induction of Active Systemic Anaphylaxis in Mice / 대한면역학회지

Human seminal plasrna (HSP) is mixture of secretion derived from various glands associated with male reproductive tract which comprises approximately 80-90% of the volume of normal ejaculate. The present study was undertaken in an effort to explore the effect of HSP pretreatment on the production of IL-1B, TNF-a and IL-12, in mice, and to investigate if HSP may cause to induce active systemic anaphylaxis (ASA) in mice. In addition, effects of HSP pretreatment on contact hypersensitivity to trinitrochlorobenzene (TNCB), antibody response to polyvinylpyrroridone (PVP), a thymus-independent antigen and on ASA induced by egg albumin (OVA) were also studied in this study. For the experiments of contact hypersensitivity, antibody response and cytokine production, mice were pretreated i.p. daily with 0.3ml of HSP or sterile saline alone (control) for 3 consecutive days before antigen sensitization or lipopolysaccharide injection for the cytokine induction. For the experiments of OVA- induced anaphylaxis, mice were pretreated by a single s.c. injection of HSP 0.3ml per mouse before sensitization. For induction of ASA in mice by HSP, a group of mice were sensitized i.p. 2 consecutive days with 0.3ml of HSP and one day with 0.3 ml of HSP plus 2x10(9) B. pertussis and 1.0 mg of alum (schedule A) or another group of mice were sensitized i.p. with a single i.p. injection of 0.3 ml of HSP with 2x10' B. pertussis and 1.0 mg of alum (schedule B). All sensitized and unsensitized control mice were challenged i.v. with 0.2ml of HSP 14 days after HSP sensitization, and mortality were observed. It was found that HSP pretreatment inhibited the production of IL-lB, TNF-a and IL-12, and also inhibited OVA-induced ASA, contact hypersensitivity to TNCB and anti-PVP antibody production. Interestingly, ASA was induced by HSP irrespective of the applied sensitization schedule. Taken together, this study may provide the direct evidences that HSP may inhibit the production of IL-1B, TNF-a and IL-12 and this may be the first to show the induction of ASA by HSP in mice.

Induction of Active Systemic Anaphylaxis and Immunological Aspects in Mice Sensitized with House Dust Mite / 대한면역학회지

We have used BALB/c mice as an animal model for the study of anaphylactic hypersensitivity to the house dust mite. For the sensitization, BALB/c mice were injected with a single dose of extracts of Oermatophagoides farinae (D. pa) or Dermatophagoides pteronyssinus (D. pt) mixed with adjuvants (aluminum hydroxide and Bordetella pertussis) intraperitonealy. On days of 15, 30, and 60 after the sensitization, the mice received a challenge dose of the same allergen intravenously to induce anaphylactic shock. The hypersensitivity reactions were scored by anaphylactic shock. And various immunological parameters, including cytokines and immunoglobulin isotypes, were studied in relation with the shock. A high level of anaphylactic shock was produced in the mice by both of the allergens, D, fa and D, pt, at 15 and 30 days after sensitization. In vitro Ag specific proliferative reponses of spleen cells from D. pt treated mice (D. pt mice) was six times higher than those from O. fa treated mice (O. fa mice). Regardless the differences in antigens, the production of IFN-r by spleen cells from D. pt mice or O. fa mice was equally high at 15 days after sensitization. However, the ability to produce IFN-r by the spleen cells from D, pt mice was three times higher compared to that from D. fa mice. The production of IL-4 by the spleen cells was enhanced slightly but not significant in both groups. In studies of the allergen-specific immunoglobulin isotypes in the sera of the mice, the level of IgE in both groups was enhanced slightly but not significant. In contrast, the level of IgG subtypes were increased in both groups. When the levels of IgG were compared by subtypes, the level of IgG1 increased significantly on day 15 when the anaphylactic shock score was maximized in both groups. Increase in IgG2a level at the day was not significant, instead, asignificant increase in IgG2 levels was observed on day 60 after sensitization when the anaphylaxis was almost discontinued. Although a higher level of IgG3 was examined on day 15 and 30 in D. pt mice and on day 60 in D, fa mice, anaphylaxis was not appeared to be associated with the levels of IgG3 in this study. The IgG1, rather than IgE, was assumed to the major factor involved in the anaphylactic response observed in this experiment. In conclusion, BALB/c mice would be an animal model for the study of anaphylactic hypersensitivity to D. fa or D, pt., which might be an essential tool for the future development of immuno-therapeutic agents.

Effects of Human Seminal Plasma on Humoral and Cellular Immune Response in Mice / 대한비뇨기과학회지

Although it has been known that human seminal plasma (HSP) suppresses immune responses, there is little data concerning in vivo effects on humoral and cellular immune responses, particularly on immediate hypersensitivity. Thus, the present study was undertaken in an effort to investigate the in vivo effect of HSP on humoral and cellular immune responses, including active systemic anaphylaxis (ASA) in mice. The immune responses investigated were delayed-type hypersensitivity (DTH) reaction to sheep red blood cell (SRBC) or human sperm antigen, hemagglutinin response, and active systemic anaphylaxis induced by egg albumin (OVA). Effect of HSP on Candida albicans infection was also studied. It was found that intraperitoneal administration of HSP before or after immunization with SRBC significantly suppressed the DTH to SRBC. HSP given after immunization with SRBC failed to suppress hemagglutinin response whereas HSP given before immunization with SRBC significantly suppressed the hemagglutinin response. Interestingly, intravaginal administration of HSP together with human sperm significantly suppressed DTH to human sperm as measured by footpad swelling reactions. HSP inhibited phagocytic function of macrophage and enhanced germ tube producing phagocytosed yeasts. Colony forming unit (CFU) of Candida albicans in kidneys of HSP-treated mice were enumerated. HSP given to mice before infection significantly increased the number of CFU in kidneys, strongly suggesting that HSP may decrease the resistance of mice to Candida infection. For the ASA experiment, mice were sensitized by i.p. injection of 500 ug OVA, 1.0 mg alum and 2x1000000000 Bordetella pertussis in 0.5 ml PBS and were challenged by i.v. inje-ction of 0.25 ml PBS containing 500 big OVA 18 days after sensitization. Surpris-ingly,HSP injection before ASA induction inhibited intensity of ASA and improved survival of anaphylaxis. Taken together, this study strongly suggests that HSP may suppress in vivo immediate and delayed immune responses and that HSP may decrease the resistance against Candida albicans infection, and this study may be the first to show the immunosuppressive effect of HSP on the induction of active systemic anaphylaxis.