EVALUATION OF THE ANTI-TUMOR EFFICACY OF TEST FORMULATION IN HUMAN BREAST CANCER XENOGRAFT TRANSPLANTED IN ATHYMIC NUDE MICE MODEL

 EVALUATION OF THE ANTI-TUMOR EFFICACY OF TEST FORMULATIONS IN HUMAN BREAST CANCER XENOGRAFT TRANSPLANTED IN ATHYMIC NUDE MICE MODEL

1.0  TEST SYSTEM DETAILS:

Species                   : Mus musculus (Mice)

Strain                     : Athymic Nude Foxn1

Age                        : 4-5 weeks

Sex                        : Female

No. of animals        : 5/Group

Total animals         : 50

 

2.0  TEST ARTICLES DETAILS

Herbo-mineral formulation containing plant extracts.

3.0  VEHICLE DETAILS

The test articles will be formulated by utilizing 0.5% methylcellulose as the vehicle.

4.0   ALLOCATION OF GROUPS:

 

Group No.	Group Description	Disease Induction procedure	Treatment administered	Dose Volume and Route
G1	Disease Control	5 × 106 GFP/Luciferase expressing MDA-MB-231 cells injected subcutaneously in the right flank in a volume of 100 µL.	0.5% MC, p.o., b.i.d.	10 ml/kg, p.o./i.p.
G2	Reference Control		Docetaxel -1 mg/kg, i.p., thrice a week + 0.5% MC, p.o., q.d.
G3	Treated with low dose		LD: 10-30 mg/kg, b.i.d.
G4	Treated with intermediate dose 1		ID-1: 30-100 mg/kg, b.i.d.
G5	Treated with high dose		HD: 100-300 mg/kg,   b.i.d.

Abbreviations: MC: Methylcellulose, p.o.-per os., i.p.-intraperitoneal,  q.d.: quaque die; bid: bis in die.

5.0  METHOD:

·         Healthy animals will be selected for the study, randomized based on body weight and will be assigned to 10 groups consisting of 10 animals each.

·         Disease control animals (assigned to group G1) will receive 0.5% MC, p.o., b.i.d.

·         Animals of group G2 will be treated with reference drug docetaxel at the dose of 1 mg/kg, i.p., three times per week.

·         Animals of group G3-G6 will be treated with HF, at different incremental dose levels as outlined in Section 4.0 of Annexure-I, twice daily.

·         ·         Green fluorescent protein (GFP)/Luciferase expressing MDA-MB-231 cells will be maintained in RPMI (Roswell Park Memorial Institute) 1640 media, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, at 37°C in a humidified atmosphere with 5% CO2 in an incubator and harvested during the logarithmic growth phase. 5 × 10⁶ cells will be subcutaneously injected into each mouse on the right flank and tumors will be allowed to develop for 14 days. Mice will then be randomly grouped based on the tumor sizes and treatment will be initiated subsequently.  

·         Tumor size and volume will be measured by utilizing vernier calipers twice a week for four weeks. Additionally, tumor progression will be monitored by in-vivo optical imaging system (IVIS, Perkin-Elmer) under transient isoflurane anaesthesia.

·         Twenty-four hours after the last administration of treatments, animals will be sacrificed under overdose of thiopentone anaesthesia. Immediately after the animal dies, the tumors will be excised and weighed. Subsequently, they will be incised in two portions. One portion will be fixed in 10% neutral buffered formalin for histopathological and immunohistochemical evaluation whereas the other will be stored at -80°C for biochemical and molecular evaluations.

 

6.0  PARAMETERS TO BE EVALUATED:

·         Body weight: Twice a week

·         Tumor size: Twice a week

·         Tumor progression by optical imaging: Twice a week

·         Western Blotting in excised tumors: Expression of cleaved Caspase-3 and Caspase 8, Bcl2, Bax, Cyclin A, p21^Cip1.

·         Histology of tumor sections stained with Hematoxylin and Eosin as well as immunohistochemistry.

 

7.0  REFERENCE(S):

1.  Jeon, Y. W. et al. Potentiation of the Anticancer Effects by Combining Docetaxel with Ku-0063794 against Triple-Negative Breast Cancer Cells. Cancer Res. Treat. 54, 157–173 (2022).

2.  Lang, S. J. et al. Antitumor activity of an Artemisia annua herbal preparation and identification of active ingredients. Phytomedicine 62, 152962 (2019).

3.  Radestock, Y., Hoang-Vu, C. & Hombach-Klonisch, S. Relaxin reduces xenograft tumour growth of human MDA-MB-231 breast cancer cells. Breast Cancer Res. 10, 1–15 (2008).

4.  Khaled, M., Belaaloui, G., Jiang, Z. Z., Zhu, X. & Zhang, L. Y. Antitumor effect of Deoxypodophyllotoxin on human breast cancer xenograft transplanted in BALB/c nude mice model. J. Infect. Chemother. 22, 692–696 (2016).

                                                         END OF THE DOCUMENT

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