Preclinical studies

RCD s.r.o.
Americká 632
252 29 Dobřichovice
DIČ: CZ47052511

EFFECT OF THE PRODUCT BIOCOL ON THE GROWTH OF HUMAN COLON CARCINOMA (LINE HCT 116) IN NUDE MICE

Principal investigator: Doc. RNDr. Pavla Poučková, CSc.
Experiment supervised by: RNDr. Marie Zadinová
Technical assistance: Eleonora Wenclová, Milena Altnerová, Olga Tachecí
Controlled by: Ing. Jiřina Polívková, CSc.
Report elaborated by: Doc. RNDr. Pavla Poučková, CSc., RNDr. Marie Zadinová
Implemented in RCD s.r.o. on 9. 6. To 18. 8. 2005

 

 

 

 

1. Target of the Study:

The purpose of the study presented here was to determine anti-cancer effect of the product BIOCOL on the growth of human colon carcinoma (line HCT 116) transplanted subcutaneously (s.c.) into the hypodermis of nude mice.


2. Material and Methods


2.1. Substance tested:

BIOCOL – the substance was delivered in the form of dark red powder. It was diluted in accordance with directions provided by the customer. The solution was prepared fresh immediately before each administration, it was diluted with drinking water and administered daily orally (p.o.) in a dose of 250 mg/kg, (5 mg/20g), in a volume of 0,2 ml/20 g (10 ml/kg) of the body weight. Animals in control groups were administered daily, (p.o.) with the same volume of drinking water (0,2 ml/20 g, 10 ml/kg of the body weight).


2.2. Tumor lines

A human tumor line of colon carcinoma (HCT-116) was used in the experiment. It was primarily acquired from the European tumor bank ECACC (European Collection of the Cell Cultures, CAMR, Salisbary, Wiltshire SP4 0JG, United Kingdom). This tumor line was multiplied in a tissue culture and nude mice were administered s.c. with 1x107 tumor cells together with 0,1 ml of Matrigel – on the right side of each the mouse.


2.3. Laboratory animals

Female CD-1 strain outbred nude mice were used in the experiment; body weight of 18 – 20 g; total of 32 animals; 16 experimental mice and 16 control mice (Supplied by: AnLab s.r.o. Praha). The mice were kept in air laminal flow boxes KAT-F/SZ-1000 (AnLab s.r.o. Praha), fed with radiation-sterilized ST-1 diet (AnLab s.r.o. Praha), and they recieved autoclaved water ad libitum.


2.4. The experimental procedure

One day before starting the administration of the therapeutic substance BIOCOL (day 0), the blood was sampled from 3 mice, from the ophthalmic plexus and the baseline blood count was established. Thereafter, 24 hours after sampling, the BIOCOL product oral administration to CD-1 was started and it continued for 10 day. The experimental animal were administered with 250 mg/kg of the product BIOCOL by gavage. On the 11th day, tumor cells were inoculated into to the right side of the nude mouse in a number of 1 x 107/mouse. After the tumor cell inoculation, the treatment with the product BIOCOL continued in the experimental group. Controls received the same volume of drinking water by gavage for the same period. The experiment was completed on the 36th day after starting the BIOCOL product oral administration.


Experimental group:
P1 – 16 outbred CD–1 strain nude mice, s.c. ca colon – line HCT 116 + p.o. 250 mg/kg of substance BIOCOL.


Control group:
K1 – 16 outbred CD–1 strain nude mice, s.c. ca colon – line HCT 116 + p.o. drinking water


The growing tumors were measured every third of fourth day and the growing tumor volume was calculated from the formula V = a x b2 x π/6, where a = tumor length, b = tumor width. The mice were also weighed every third or fourth day. The experiment was completed on the 36th day after starting the treatment.


2.5. The experiment evaluation

The evaluation of the experiment was carried out based on comparing tumor growth curves in experimental animals in comparison with controls. In the course of the experiment and at the end of the experiment, percent tumor growth inhibition – %TGI – was calculated based on the tumor average volume (%TGI = 1 –(average tumor volume in experimental group/average tumor volume in control group)) x 100. The results obtained were statistically evaluated by the non-parametric two-sided Student test. In the course of the experiment and at the en of the experiment, photographic documentation of the therapy efficacy was provided in comparison with controls.


3. Results and Discussion

The results are summarized in Tables 1 to 11 and Graphs 1 to 9.

Experimental group: 16 outbred CD-1 strain nude mice, s.c. ca colon – line HCT 116 + p.o. 250 mg/kg of the substance BIOCOL.

Control group: 16 outbred CD-1 strain nude mice, s.c. ca colon – line HCT 116 + drinking water.


The product BIOCOL, administered for 10 days before the s.c. Inoculation of tumor cells and subsequently also daily after this inoculation, prolonged statistically significantly the c.a. colon (HCT 116) growth latency period in experimental nude mice, in average by 7.93 day – to 18.56 days (by 74%), i. e. to 174.6%, in comparison with the control – untreated group, in which the tumor growth was observed after 10.63 days (100%) (Tables 1, 2, Graphs 1, 2).


The product BIOCOL partially inhibited the growth of the human colon carcinoma (line HCT-116) on nude mice. The inhibition achieved was statistically significant from the 18th day to the 36th day of the experiment at a 1% significance level. The achieved percent tumor growth inhibition against controls (%TGI) was of level. The achieved percent tumor growth inhibition against controls (%TGI) was of 64.4% on the 18th day of the experiment, 56.1% on the 22nd day of the experiment, 53.8% on the 25th day of the experiment, 47.04% on the 29th day of the experiment, 39.53% on the 32th day of the experiment and 31.61% on the 36th day of the experiment. (Table 3, 4, 5, 6, 7, Graphs 3, 4, 5, 6).


In the case of considering effects of the product BIOCOL on the nude mice body weight, the dose of 250 mg/kg induced body weight decrements in experimental nude mice and, after the 5th week of administration, also pink to red color of the nude mice skin. The nude mice body weight decrements were statistically significant at a 1% significance level from the 11th day to the 36th day of the therapy (Tables 8, 9,10, 11 Graphs 7, 8,9).


From the results, it is obvious that the product BIOCOL partially inhibited the growth of the human colon carcinoma in nude mice, and that the effect of the preventive administration of the product from the first day of the experiment, before the tumour cell transplantation, was particularly beneficial. With increasing time of the product administration, the percent tumour growth inhibition stepwise decreased. With respect to the general appearance of the mice we assume that there was an overdose with the product administered (see the photo).


4. Conslusion

• The preventive administration of the product BIOCOL for 10 days before the Subcutaneous inoculation of tumor cells (with subsequent daily administration after the inoculation), prolonged statistically significantly the latency period of the s. c. growth of colon carcinoma (line HCT-116) in experimental nude mice in comparison with controls - by 74% (by 7.93 days).
  
  
• The product BIOCOL exerted a statistically significant inhibiting effect on the s. c. growth of the human colon carcinoma (line HCT-116) in CD-I strain nude mice. On the 18th day of the experiment, a 64.4% tumor growth inhibition was achieved in comparison with controls. 
  
  
• With increasing time of administering the product BIOCOL, the percent tumor growth inhibition was stepwise reduced; on the 36th day, there was a 31.61% tumor growth inhibition in comparison with controls.
  
  
• The product BIOCOL, in its administered daily oral dose of 250 mg/kg, reduced statistically significantly the body weight in experimental mice.

In Prague on 15. 9. 2005


  
Doc. RNDr. Pavla Poučková, CSc.

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