Evaluation Of Toxicological Profile (Acute And Sub Acute...

G.V.N.Kiranmayi., PharmaBitika.,2014.1(1),094-101

PharmaBiotika/2014/1/1/094-101-©2014www.pharmaBiotikaworld.com All rights reserved. 94

Research Article ISSN 2408-9702

Evaluation Of Toxicological Profile (Acute And Sub

Acute) Of Cremophor in Dacarbazine Formulation in Rodents

Dr.K.Ravishankar [1], G.V.N.Kiranmayi *[1], P.Priyabandhavi [1], D.Peer Basha [2]

1. Sri Sai Aditya Institute of Pharmaceutical Sciences and Research. 2. Therdose Pharma Pvt. Ltd., Hyderabad

Abstract Cremophor is widely used as a solvent in many cancer formulations, however, there are no complete toxicity studies done on it for its use as a solvent in Dacarbazine formulation. The present investigation was conducted to determine the safety of cremophor as a solvent in Dacarbazine formulation by determining its potential toxicity after acute and subacute study in rodents. In acute study, The mice were divided into three Groups of six animals and each group received once 100, 500, 1000 mg/kg dose of formulation by intravenous route .In the sub acute study, the first group of rats served as the control receiving standard dacarbazine while second Group was given vehicle and the other three Groups were given solvent containing formulation at doses of 25, 50 and 100mg/kg respectively via intravenous route every 5 days up to 28 days. Behavioural changes, body weight were determined. Biochemistry and haematology analysis were done for subacute toxicity study. Histopathological examination of rat kidney, liver and brain were carried after 28 days. The IV LD50 for the, formulation was found to be 466mg/kg.subacute toxicity showed no significant changes in biochemical and haematological parameters. No pathological changes were observed in rat liver, kidney and brain. The biochemical, haematological and histopathological parameters indicates the use of cremophor as a solvent in dacarbazine formulation. Based on the observed findings the Cremophor in Dacarbazine formulation was fairly nontoxic at the IV dose tested, as it did not cause any death or adverse behavioural changes and Cremophor can be incorporated in Dacarbazine formulation. Key words: Cremophor, Dacarbazine, Acute, Subacute. INTRODUCTION Dacarbazine is a non-classical Alkylating agent that causes DNA mispairing and strand breakage, leading to cell death (necrosis). Its exact mechanism is not completely understood. It is a cell cycle nonspecific drug, meaning that it causes cell damage and death throughout the life cycle of a cell, and not at any one particular time. Dacarbazine is commonly used as a single agent in the treatment of metastatic melanoma, and as part of chemotherapy regimen to treat Hodgkin lymphoma, and sarcoma.

Cremophor EL is a pale yellow, oily liquid, non-ionic solubilizer and emulsifier obtained by causing ethylene oxide to react with castor oil. Cremophor is a formulation vehicle used for various poorly-water soluble drugs, including the anticancer agent paclitaxel (Taxol). The pharmacokinetic behavior of CrEL is dose-independent, although its clearance is highly influenced by duration of the infusion. This is particularly important since CrEL can affect the disposition of various drugs by changing the unbound drug concentration through micellar encapsulation. In addition, it has been shown that CrEL, as an integral component of paclitaxel chemotherapy, modifies the toxicity profile of certain anticancer agents given concomitantly, by mechanisms other than kinetic




interference. The fine degree of dispersion resulting from addition of Cremophor EL allows a drug to be absorbed more readily and increases its efficiency. Cremophor EL promotes the penetration of a number of active substances and exerts better effect. Cremophor EL is an excipient or additive in drugs. Therapeutically, modern drugs are rarely given in a pure chemical state, so most active ingredients are combined with excipients or additives such as Cremophor EL. In the present study Cremophor has been used as a solvent in Dacarbazine formulation because so far its use as a solvent in Dacarbazine formulation has not yet been reported, if the solvent passes the toxicity tests there is a further scope for use of this solvent in different formulations as an solvent. MATERIALS AND METHODS Experimental Animals Wistar albino rats (180-250gm) and Wistar albino mice (25-30gm) were procured from National Institute of Nutrition (NIN), Hyderabad. All the animals were kept under standard environmental condition (25±2°C) and relative humidity 44-56%, with a dark and light cycle of 12±1h. The animals had free access to water and standard diet. Rats and mice were deprived of food but not water (16-18) h prior to administration of the formulation. The protocol of the experiment was approved by Institutional Animal Ethical Committee as per the guidance of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Chemicals Dacarbazine was purchased from Aadi pharmaceuticals; Cremophor was purchased from Nikunj Chemical Limited. Acute Toxicity For acute study, The mice were divided into 3 groups of 6 animals and each group received once 100, 500, 1000 mg/kg dose of formulation by intra venous route for 1 d. Control received standard drug at the same volume. Observations were made and recorded systemically 1, 2, 4 and 24h. After substance administration the visual observation included skin changes, morbidity, aggressively, sensitivity of sound and pain, as well as respiratory movement. The number of survivors was noted after 24 h. The LD50 was then determined at the end of the experiment based on Miller and Tainter method. Sub Acute Toxicity In the sub acute study, Animals were divided into five groups containing 6 animals randomly. Group – I was fed with dacarbazine reference standard every five days for up to 28days acting as control, Group- II was given only vehicle. Group – III, IV, V were given 25, 50,100mg/kg doses of solvent containing formulation every 5 days up to 28 days. The toxic manifestation such as body weight, mortality and food and water intake (data not shown) was monitored. After 28 d all surviving animals were fasted overnight. In the mean time day 0,14,28 The heparinised blood samples were collected for determining haematological parameters and the serum from non-heparinised blood was carefully collected for determining clinical blood chemistry. Rats were anesthetized with ether on day 28. Animals were sacrificed after blood collection the internal organs were weighed to determine relative organs weights and observed for gross lesions. The internal organs were preserved in 10 % buffered formaldehyde solution for histological examination. Biochemical estimations Blood collected into non-heparinised tubes were then centrifuged at 3000 rpm for 10 min. The serum was used for the estimation of biochemical parameters to like Serum glutamic oxaloacetic transaminase (SGOT), Serum glutamic pyruvic transaminase (SGPT), Alkalinephosphatase (ALP), cholesterol, glucose, creatinine, albumin and total protein were analyzed by auto-biochemistry analyzer using standard ROBONIK laboratory kits.




Haematological assay Blood sample collected in the heparinised tubes were used to investigate Red Blood Cells (RBC), White Blood Cells (WBC), Mean Cell Haemoglobin (MCH), Mean Corpuscular Volume (MCV), Haemoglobin (HGB), Mean Corpuscular Haemoglobin Concentration (MCHC), Hematocrit (HCT), Platelet Counts (PLT) using Blood Cell Counter. Histopathological analysis. Rats were perfused with buffered (0.4 M phosphate buffer, pH 7.6) 4% paraformaldehyde. The brain, liver, kidneys were removed from each animal and immersion-fixed in the same fixative for 24 h at room temperature. Tissue sections (4 μm) were prepared after dehydration and were embedded in paraffin. The sections were stained with haematoxylin and eosin (H&E) and subsequently processed for histopathological examination under light microscope.

List of organs subjected for histopathological examination were: Brain*, Liver*, Kidneys*, Statistical Analysis: All findings such as clinical signs of toxicity, body weight changes, haematology and blood chemistry were tabulated. Data on all parameters were evaluated by one-way analysis of variance (ANOVA) with post test, Dunnett Multiple Comparison test using statistical software (Graph pad Prism, US) with p<0.001

RESULT AND DISCUSSION In acute toxicity study oral administration of the formulation doses from 100 to 1000 mg/kg did not produce significant changes in behaviours, breathing, cutaneous effects, sensory, nervous systems, responses and gastrointestinal effects in mice. These effects were observed during the experimental period 24 h. Death of mice was noted at 500 and 1000 mg/kg. The LD50 value obtained was 466mg/kg body weight. The rats in subacute toxicity study received formulation in the doses of 25,50,100mg/kg body weight every 5 days up to 28 days did not result in death of the animal. No sign of observable toxicity was detected during the experimental period according to the OECD guideline. Generally, the change in body weight gain and internal organ weights is a simple and sensitive index of toxicity after exposure to toxic substances. In subacute toxicity study rats treated with 25, 50,100mg/kg doses of formulation had a progressive weight in body and organ gained. The increase in weight was not significantly different from that of the control. The progressive increase in body weight and organ weight at dose of 25,50,100mg/kg of rats during 28 days of administration of cremophor containing formulation may indicate the improvement the nutritional state of the animal. The growth response effect could be as a result of increased food and water intake. The calculated relative weight of the control and treated animal groups varied from one organ to other, no significant differences were noted in the relative weight of other organs (liver, heart, lung, spleen and kidney). In the sub acute toxicity study, the vehicle & formulation test groups did not show any significant changes in body weight increment compared to the control group in and rats (Table 1). Since, the changes in body weight have been used as an indicator of adverse effects of drugs and chemicals. Analysis of blood parameters is relevant to risk evaluation as the changes in haematological system have a higher predictive value for human toxicity, when data are translated from animal studies. The haematological and biochemical parameters (hepatic and renal function tests) (Tables 2, 3) did not show any significant changes in the vehicle, test groups when compared to the control group in rats.




The histopathological section of various organs such as the liver, kidney, brain, revealed normal architecture on comparison with the control group in rats (fig 1, 2, 3 .No abnormality was detected in other organs indicating that the test drug treatment did not cause any abnormality in other organs under gross examination .In rats treated with Vehicle and formulation did not reveal any significant constant histopathological lesions.

There were no significant changes in any liver function parameters, such as SGPT, SGOT ALP compared to the control group. Increase in these parameters would have indicated hepatocyte damage (Aniagu, et al.,2005 )& represent liver impairment, since these are important indices of liver toxicity (Hayes. A. W , 2006) . Serum cholesterol and proteins are mainly regulated via synthesis in the liver and increase or decrease in serum concentrations of constituents suggests some alterations in liver functions. As these enzymes and biochemical parameters were not altered by administration of cremophor and cremophor containing formulation indicating that there may be no liver damage.

Renal functional markers like creatinine plasma levels(Ganong ,1977 )remained normal after administration, this indicates that the test drug did not interfere with renal function and that renal integrity was preserved. Also, there were no significant changes regarding various haematological parameters such as Hb, RBC, WBC,HCT,MCV and MCH which indicates that Both Vehicle And Test Formulation may not be toxic and does not affect circulating red cells haematopoiesis, or leucopoiesis. Haematopoiesis and leucopoiesis were also not affected even though the haematopoietic system is one of the most sensitive targets for toxic compounds and an important index of physiological and pathological status in man and animals (Adeneye.A.A., et al., 2006) Conclusion: The Present Findings Suggest That cremophor is nontoxic since no marked changes in haematological, biochemical, and histopathological parameters were observed in rats . Thus, cremophor is considered to be safe vehicle in Dacarbazine formulation.

Table 1: Effect On Mean Body Weight Of Rats ( g ) (Mean ± SEM)

Group Day 1 Day 3 Day 9 Day 14 Day28 I

Male 184.1±6.45 190.1±9.67 214.2±10.45 223.4±9.43 225.6±8.82 Female 175.3±6.65 182.1±8.24 185.6±11.76 182.7±11.35 183.6±10.98

II

Male 184.5±6.49 186.9±6.53 203.8±6.78 213.9±6.57 220.9±6.37 Female 175.8±6.41 173.1±12.41 189.6±8.94 185.3±9.39 183.9±10.98

III

Male 182.5±7.63 190.9±6.82 214.4±8.82 227.2±10.08 239.2±10.73

Female 178.4±12.08 182.3±12.94 186.7±11.76 181±12.906 180±11.80

IV

Male 182.8±7.22 180.7±8.78 204.3±8.33 211.2±7.14 223.6±8.29

Female 174.6±7.71 181.2±6.69 185±9.88 181.4±10.04 182±9.92

V

Male 183.4±7.10 182.3±8.73 195±10.41 204.7±10.08 210±8.53 Female 174.6±7.10 178.1±9.39 184.9±9.43 181±9.14 180.6±9.76

n = 6; Vehicle, test groups differs insignificantly from control




Table 2: Haematological data of Wistar rats (Mean ± SEM) GROUP RBC

(x 106 /ul) MCV

(fl) HCT (%)

Platelet Count

(x 103 /uL))

WBC (x 103 /uL)

Hb (g %)

MCH (pg)

MCHC (g/d

l) Day 0

I Male 7.2±0.20 55.5±0.57 39.9±1.06 436±19.35 17.9±2.90 12.9±0.24 18.2±0.20 32.8±0.12 Female 7.6±0.06 51.9±1.18 38.7±0.53 524.8±18.45 10±2.00 14.9±0.04 19.7±0.12 38.6±0.45

II Male 7.2±0.08 54±0.94 38.8±0.82 448.3±23.47 16±2.73 12.7±0.24 17.7±0.24 32.8±0.12 Female 7.44 ±0.16 49.7±0.49 37±0.69 475.7±25.31 12.3±2.86 14.5±0.24 19.5±0.24 39.1±0.16

III

Male 6.5±0.29 55.6±0.86 35.7±1.47 385.8±19.18 12.4±0.94 11.6±0.53 18.2±0.33 32.7±0.20 Female 7.66±0.15 50.5±0.57 38.7±0.82 473.3±23.10 10.2±0.61 14.8±0.29 19.3±0.16 38.1±0.45

IV

Male 6.7±0.20 55.2±0.73 35.9±1.31 384.7±23.88 11.7±0.98 11.8±0.41 18.3±0.24 33.1±0.16 Female 7.67±0.09 51±0.65 39.1±0.53 477.7±16.65 13.5±1.84 14.9±0.16 19.4±0.20 38.1±0.29

V

Male 7.1±0.24 55.3±1.18 39±1.06 429.5±14.37 16±2.86 12.9±0.33 18.2±0.37 33.1±0.12 Female 7.46±0.14 50.1±0.20 37.4±0.69 495±24.12 9.6±2.04 14.4±0.24 19.3±0.16 38.6±0.20

Day 14 I Male 7.48±0.17 52.9±0.57 39.6±1.27 507±15.43 18.6±0.86 14.9±0.33 20±0.20 37.8±0.53

Female 7.97±0.09 50.8±0.41 40.5±0.33 515.7±18.73 12.3±1.71 15.4±0.16 19.3±0.16 38±0.12 II Male 7.2±0.32 53±1.22 38±1.06 486.2±34.16 17.8±2.37 14.2±0.45 19.8±0.41 37.4±0.37

Female 7.75±0.18 50.9±0.41 39.5±0.65 550.7±24.73 13.1±1.18 15.1±0.24 19.5±0.2 38.2±0.20

III Male 7.06±0.26 54.3±1.02 38.2±0.73 545.3±29.80 13.1±1.76 14.4±0.37 20.5±0.33 37.7±0.33

Female 8.16±0.22 50.2±0.29 40.9±1.02 510.8±22.53 11.8±0.37 15.6±0.37 19.2±0.20 38.2±0.29

IV Male 7.29±0.12 52.4±0.41 38.2±0.61 531.8±21.14 16.6±2.41 14.3±0.20 19.7±0.12 37.7±0.33

Female 8.03±0.18 50.2±0.61 40.3±0.49 487±11.27 11.9±1.14 15.4±0.16 19.2±0.24 38.2±0.20

V Male 7.61±0.28 51.4±0.94 39.2±0.78 512.3±29.43 17.3±1.92 14.4±0.29 19.3±0.33 36.8±0.20

Female 8.26±0.15 50±0.29 41.3±0.78 530.2±23.63 13±0.65 15.8±0.33 19.1±0.12 38.1±0.08 Day 28

I Male 7.59±0.18 51.5±0.57 39.8±1.14 548.2±17.42 19.3±1.06 14.95±0.24 19.92±0.16 38.3±0.33 Female 7.99±0.07 50.98±0.82 41.2±0.39 512.7±18.61 14.2±1.84 16.2±0.12 19.5±0.14 38.3±0.2

II Male 7.2±0.28 52±1.06 38±0.90 492.8±26.65 18.9±2.53 14.82±0.31 19.92±0.27 38.5±0.24 Female 7.79±0.16 51.2±0.39 40.2±0.61 570.4±25.02 14.2±2.12 15.5±0.24 19.5±0.22 38±0.18

III

Male 7.12±0.28 53.8±0.94 39.6±0.49 542.2±23.80 14.3±1.31 15.9±0.49 20.3±0.31 38.4±0.27 Female 8.2±0.18 50.1±0.27 41.2±0.98 520.3±22.16 12.3±0.39 16.8±0.33 19.4±0.16 38.3±0.33

IV

Male 7.25±0.14 51.8±1.10 39.7±1.14 540.3±24.45 17.8±1.71 15.5±0.24 20.5±0.22 38.6±0.24 Female 8.18±0.14 50.35±0.65 40.5±0.51 497±12.33 13.01±1.43 15.8±0.16 19.4±0.20 38.2±0.27

V

Male 7.59±0.24 50.8±2.7 39.6±0.73 543.6±22.86 18.6±2.16 15.2±0.29 19.25±0.34 37.9±0.18 Female 8.32±0.14 50.2±0.27 41.6±0.73 545±23.88 14.2±1.31 16.6±0.29 19.2±0.16 38.25±0.12

n = 6 ; Vehicle , test groups differs insignificantly from control.




Table 3: Biochemical Data of Female Wistar Rats (Mean ± SEM) GROUP GLUC

(mg/dl) Cr

(mg/dL) SGOT (IU/L)

ALB (mg/dl)

ALKP (IU/L)

CHOL ( mg/dl)

SGPT (IU/L)

Tp (mg/dl)

Day 0 I Male 82.2±2.24 0.5±0.04 41.17±25.32 4.1±0.04 107.5±6.94 68.5±4.49 34.8±1.18 7±0.16 Female 75.1±1.43 0.2±0.04 35.1±11.27 3.8±0.12 135.8±2.57 57.6±4.45 36.2±3.55 6.3±0.16

II Male 81.7±2.49 0.5±0.04 45.28±3.12 4.1±0.08 111.1±8.16 61.2±3.88 34.7±0.78 7±0.20 Female 75±2.16 0.3±0.00 30.1±5.55 3.7±0.12 126.9±6.86 51.5±1.96 33.1±2.49 6.6±0.20

III

Male 83.5±2.37 0.4±0.04 45.69±0.05 4.1±0.08 122.2±5.43 64.7±4.78 34.1±1.06 7.1±0.16

Female 80.3±3.14 0.3±0.04 39.9±6.78 3.7±0.04 128.3±6.08 52.8±2.45 33±3.43 6.3±0.16

IV Male 76.3±2.00 0.4±0.04 50.87±11.23 4.2±0.04 116.3±6.69 59±3.39 34.3±2.41 7.3±0.33

Female 77.2±1.96 0.3±0.04 41.3±5.31 3.8±0.12 127±4.86 49±2.20 30.2±2.24 6.7±0.12

V Male 79.6±4.20 0.5±0.04 49.93±8.59 4.1±0.12 103.4±8.04 58±2.24 33.7±2.82 6.9±0.20

Female 81.6±2.98 0.3±0.04 42.6±6.78 3.8±0.16 130.2±4.73 56.2±5.06 30.7±2.45 6.6±0.37 Day 14

I Male 86.8±3.10 0.4±0.04 42.63±55.40 4±0.08 92.2±11.84 66.7±3.96 35.8±1.14 8±0.12

Female 90.4±6.37 0.3±0.04 36.3±6.29 3.8±0.12 123.1±5.96 53.2±2.69 32±2.86 6.6±0.12

II Male 90.4±3.55 0.5±0.00 45.13±25.69 4±0.04 90.7±7.39 58.3±4.12 36.6±1.31 8.5±0.41

Female 85.7±4.90 0.3±0.04 34.8±9.51 3.8±0.08 125±1.35 52.7±1.18 30.5±2.04 6.4±0.20

III

Male

81.8±3.22 0.5±0.04 34.50±31.36 3.8±0.12 91.8±5.14 63.7±4.73 35.7±3.51 7.8±0.33

Female 76±1.55 0.3±0.04 37.8±8.94 3.6±0.08 123.7±3.71 52±1.55 32.2±1.88 6.4±0.16

IV Male 93.7±7.67 0.5±0.04 33.97±1.75 4±0.08 92.8±5.88 63±5.51 33.7±2.49 7.6±0.16

Female 73.5±2.94* 0.3±0.00 40.6±3.35 3.5±0.08 129.7±4.90 52.2±1.96 31.2±2.33 6.2±0.12

V Male 81.6±4.00 0.5±0.04 33.97±4.02 4±0.12 97.9±7.59 64.8±4.00 40.4±2.61 7.8±0.24

Female 74.5±3.31* 0.3±0.04 42.5±10.37 3.7±0.08 111.3±7.43 59.7±2.49 28.2±1.63 6.2±0.29 Day 28

I Male 86.7±2.57 0.5±0.04 42.9±6.24 4.1±0.04 90.5±8.29 67.8±4.45 36.3±1.18 8.5±0.12 Female 80.6±2.12 0.3±0.04 38.9±8.34 3.8±0.12 120.6±5.02 52.3±3.35 34.4±3.35 6.8±0.14

II Male 91.7±2.82 0.4±0.04 43.3±8.49 4.2±0.08 90.2±7.84 56.9±4.12 38.2±1.18 8.8±0.20 Female 88.3±4.00 0.3±0.04 40.3±6.243 3.6±0.08 120.4±1.84 53.2±1.43 29.2±2.24 6.6±0.20

III

Male

80.9±2.57 0.5±0.04 45.3±6.45 3.9±0.13 90.8±5.06 62.3±4.69 36.3±1.76 8.1±0.24

Female 75±1.59 0.3±0.04 41.2±8.29 3.5±0.08 132.3±5.39 51.8±2.12 33±2.78 6.5±0.16

IV Male 85.4±6.24 0.5±0.04 44.2±5.84 4.1±0.04 90.3±6.82 64.8±4.16 32.9±2.37 7.8±0.24

Female 72.2±2.16 0.3±0.04 42.3±4.16 3.6±0.08 131. 3±4.82

53.6±1.92 32.2±2.29 6.1±0.12*

Male 82.3±4.04 0.4±0.04 38.8±7.43 4±0.08 94.3±6.65 66.3±3.35 40.8±2.78 8.2±0.20




V Female 73.5±3.06 0.2±0.04 43.2±8.33 3.7±0.14 108.3±5.92 62.3±3.39* 30.3±2.04 6±0.27* n = 6 ; *significantly different from the control at p<0.001

Fig 1: Histopathological images of Rat liver

Control

Vehicle

High dose

Fig 2 : Histopathological images of Rat brain

Control( 10 x ) vehicle( 10 x ) High dose( 10 x )




Fig 3: Histopathological images of Rat kidney

References

1. Aniagu, S. O., Nwinyi, F. C., Akumka, D. D Ajoku, L. A., Dzanma, S. Lzebe, K. S., Ditse, M. Nwaneri, P. E. C., Wambebe, C. and Lamaniel, K(2005).Toxicity studies in rats fed nature cure bit ters Afr. J. Biotechnol 4 ,72–78.

2. Ganong. W. F; Review of Medical Physiology(1977;); Lange Medical Publications; California., 8, 136-137.

3. Hayes. A. W(2006); Principles and Methods of Toxicity (Guidelines for acute oral toxicity testing) New York: Raven Press Ltd., 184.

4. Adeneye.A.A., O.P.Ajagbonna., T.I. Adeleke., et al,( 2006); Journal. Ethnopharmacol,105, 374–379.

Control (50x )

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High dose(50x )

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