Acute and Subacute Toxicity study of Milnacipran Hydrochloride in Wistar rats by Oral Route

D. Benito Johnson*, R. Suresh, Prakash Rao Prathima, R. Venkatnarayanan and Ashir Ahammad P.M.

Department of Pharmacology, R.V.S. College of Pharmaceutical Sciences, Sulur, Coimbatore, Tamil Nadu

ABSTRACT:

Toxicity of a substance is nothing but unwanted or series of adverse events that was initiated after administration of particular chemical, physical or biological agent. Acute toxicity study aim is to determine the occurred toxic manifestation of the administered test substance after expose to animals in one or more doses for a period of 14 days. The study provides the information or determination of therapeutic index, i.e. T.I. = LD50/ ED 50. Sub-acute toxicity testing evaluates the toxic effects of drug on repeated exposure and also provides the information on delayed and cumulative effect of the chemicals on the tissues or other biochemical mechanisms Depression is one of the most common psychiatric disorders. The symptoms of depression are often subtle and unrecognized both by patients and by physicians. Major depression remains difficult to treat, despite the wide array of registered antidepressant. Milnacipran is indicated for the treatment of major depressive disorder and management of fibromyalgia. Milnacipran inhibits norepinephrine and serotonin reuptake in a 3:1 ratio, in practical use this means a balanced (equal) action upon both transmitter.

KEYWORDS: Milancipran, Depression, Acute toxicity, Sub-acute toxicity

INTRODUCTION:

Depression is one of the most common psychiatric disorders. At any given moment, about 3-5% of the population is depressed (point prevalence), and an estimated 10% of people may become depressed during their lives (lifetime prevalence). The symptoms of depression are often subtle and unrecognized both by patients and by physicians^{1, 2}. Patients with vague complaints that resist explanation as manifestations of somatic disorders and those who might be simplistically described as "neurotic" should be suspected of being depressed. When a person has a depressive disorder, it interferes with daily life, normal function and causes pain for both the person with the disorder and those who care about him or her ^{3, 4}. Depression is a common but serious illness, and most that experience it need treatment to get better. Many people with a depressive illness never seek treatment. But the vast majority, even those with the most severe depression, can get better with treatment. Intensive research into the illness has resulted in the development of medications, psychotherapies, and other methods to treat people with this disabling disorder ^{5, 6}. Depression, as a transient mood, is experienced by everyone including the early. It is a self limiting and dose doesn’t need any medical intervention. Depressive illness, however, is a different matter. It is a serious, debilitating, usually recurrent, sometimes chronic illness with considerable morbidity and mortality. Evidence suggests that even patients with mild clinical depression suffer impaired social and occupational functioning, apart from distress. About 15 % depressed patients commit suicide, accompanied by a high mortality due to medical causes^{7, 8}.

Fig 1

Antidepressants work to normalize naturally occurring brain chemicals called neurotransmitters, notably serotonin and Norepinephrine. Other antidepressants work on the neurotransmitter dopamine. The newest and most popular types of antidepressant medications are called selective serotonin reuptake inhibitors (SSRIs), serotonin Norepinephrine reuptake inhibitors (SNRIs) ⁹.

Serotonin and Norepinephrine, dopamine is thought to be an important neurotransmitter in the pathophysiology of depression. The pharmacological evidence does not enables a clear distinction to be drawn between the Noradrenaline and 5HT theories of depression. Plasma Noradrenaline actually tends to be higher in depressed than in normal subjects, possibly because it reflects peripheral sympathetic activity, which increases with the anxiety that often accompanies depression (Bipolar depression)¹⁰.

Clinically, it seems that inhibitors of Noradrenaline reuptake are equally effective as antidepressant though individual patients may respond better to one or the other. Various Pharmacological evidence supporting the monoamine hypothesis of depression by blocking Noradrenaline and 5- HT reuptake, increase store of Noraderenaline and 5-HT, inhibit Noraderenaline synthesis, increase 5-HT synthesis, increases CNS response to Noraderenaline and 5-HT¹¹.

Simplified diagram (Fig 1) showing mechanisms believed to be involved in the pathophysiology of depression¹².

Common signs and symptoms of depression ¹³

Ø Emotional symptoms

Misery, apathy and pessimism

§ Low self –esteem: feeling of guilt, inadequacy and ugliness

§ Indecisiveness, loss of motivation.

Ø Biological symptoms

§ Retardation of thought and action

§ Loss of libido

§ Sleep disturbance and loss of appetite

MATERIALS AND METHODS:

TEST ITEM

Ø Milnacipran hydrochloride

TEST ITEM PREPARATION

Ø The test item for administration is in standard volume of 2ml/kg orally

Concentrations

Ø Low dose–4.5mg/ml, middle dose–11.5mg/ml and high dose–22.5mg/ml

Table - 1

S. No.	No. of Animals/Sex	Milnacipran hydrochloride (mg/kg/day)	Duration of treatment	Study Period
1.	10 (5M + 5F)	45	single dose	14 days
2.	12 (6M + 6F)	0 (VC)	28 days	28 days
3.	12 (6M + 6F)	9 (LD)	28 days	28 days
4.	12 (6M + 6F)	23 (MD)	28 days	28 days
5	12 (6M + 6F)	45 (HD)	28 days	28 days

Vehicle Control (VC) - No exposure to test item (Water for injection)

Low Dose (LD) - Equivalent to recommended human dose

Middle Dose (MD) - 2.5 times of recommended human dose

High Dose (HD) - 5 times of recommended human dose

TEST SYSTEM

Age & weight of animals

Ø Wistar rats 6-8 weeks of age and weighing about 120-180 gm of both sex has been used

Source of animals

Ø The animals used in this study were obtained from Uni-Sankyo Ltd., Hyderabad

(Reg. No. 93/1999/CPCSEA)

Rationale for the selection of wistar rats as test system

Ø Wistar rats are one of the recommended species as test system for the conduct of toxicity studies. They have been widely used throughout the Industry for the evaluation of product safety. Extensive data available for comparative purpose.

Route of administration and reason for choice

Ø Oral route has been chosen because it is one of the proposed routes for administration to humans. The dosage can be accurately administered through oral route. The oral route is one of the proposed routes for toxicity testing.

RANDOMIZATION

Ø Randomization ensures the allocation of treatments to animals/groups was independent of their characteristics and was similar in all the groups. It was also taken care while randomization, base variables were homogenized and were allotted to different groups. The need for randomization applies not only to the allocation of the animals to the different (control as well as treatment) groups but also to anything that can materially affect the recorded response. The data shown in Table – 1.

Identification of animals

Ø Animals were identified by tail marking with picric acid. Each cage identified with individual cage tags by study number, animal number, dose, group, species, sex and date of initiation.

STASTICAL ANALYSIS

The data on food intake, body weight, hematology, biochemistry and various organ weights were subjected for analysis using SAS system 8.2. The difference between the groups is tested.

RESULTS AND DISCUSSION:

Table – 2:Acute toxicity study Pre terminal death(S) table

Name of the test item: Milnacipran hydrochloride

Dose: 45 mg/kg (5 X TD)

S.No	Days of observation	ANIMALS
		M	F	M	F	M	F	M	F
1	1	0/3	0/2	0/3	0/2	0/3	0/2	0/3	0/2
2	2	0/3	0/2	0/3	0/2	0/3	0/2	0/3	0/2
3	3	0/3	0/2	0/3	0/2	0/3	0/2	0/3	0/2
4	4	0/3	0/2	0/3	0/2	0/3	0/2	0/3	0/2
5	5	0/3	0/2	0/3	0/2	0/3	0/2	0/3	0/2
6	6	0/3	0/2	0/3	0/2	0/3	0/2	0/3	0/2
7	7	0/3	0/2	0/3	0/2	0/3	0/2	0/3	0/2
8	8	0/3	0/2	0/3	0/2	0/3	0/2	0/3	0/2
9	9	0/3	0/2	0/3	0/2	0/3	0/2	0/3	0/2
10	10	0/3	0/2	0/3	0/2	0/3	0/2	0/3	0/2
11	11	0/3	0/2	0/3	0/2	0/3	0/2	0/3	0/2
12	12	0/3	0/2	0/3	0/2	0/3	0/2	0/3	0/2
13	13	0/3	0/2	0/3	0/2	0/3	0/2	0/3	0/2
14	14	0/3	0/2	0/3	0/2	0/3	0/2	0/3	0/2
15	15	0/3	0/2	0/3	0/2	0/3	0/2	0/3	0/2

Table – 3: Home cage observations

Name of the Test item: Milnacipran hydrochloride

Animal ID	Days	Body posture (%)		Respiration character (%)		Tremors
		Normal	Abnormal	Normal	Abnormal	None
01,02,03	1st Day	100(3)	Nil	100(3)	Nil	100(3)
	8th Day	100(3)	Nil	100(3)	Nil	100(3)
	14th Day	100(3)	Nil	100(3)	Nil	100(3)
04,05	1st Day	100(2)	Nil	100(2)	Nil	100(2)
	8th Day	100(2)	Nil	100(2)	Nil	100(2)
	14th Day	100(2)	Nil	100(2)	Nil	100(2)
06,07,08	1st Day	100(3)	Nil	100(3)	Nil	100(3)
	8th Day	100(3)	Nil	100(3)	Nil	100(3)
	14th Day	100(3)	Nil	100(3)	Nil	100(3)
09,10	1st Day	100(2)	Nil	100(2)	Nil	100(2)
	8th Day	100(2)	Nil	100(2)	Nil	100(2)
	14th Day	100(2)	Nil	100(2)	Nil	100(2)

Table – 4: Open field activity

Name of the test item: Milnacipran hydrochloride, Dose: 45 mg/kg (5 X TD)

Animal ID	Days	No. of rearings	Gait	Tail elevation
		Normal	Normal	Lifted while walking	Occasionally lifted
1,2,3,	1st Day	100(3)	100(3)	100(3)	Nil
	8th Day	100(3)	100(3)	100(3)	Nil
	15th Day	100(3)	100(3)	100(3)	Nil
4,5	1st Day	100(2)	100(2)	100(2)	Nil
	8th Day	100(2)	100(2)	100(2)	Nil
	15th Day	100(2)	100(2)	100(2)	Nil
6,7,8	1st Day	100(3)	100(3)	100(3)	Nil
	8th Day	100(3)	100(3)	100(3)	Nil
	15th Day	100(3)	100(3)	100(3)	Nil
9,10	1st Day	100(2)	100(2)	100(2)	Nil
	8th Day	100(2)	100(2)	100(2)	Nil
	15th Day	100(2)	100(2)	100(2)	Nil

Table – 5 OPEN FIELD ACTIVITY (Contd.)

Name of the test item: Milnacipran hydrochloride, Dose: 45 mg/kg (5 X TD)

Animal ID	Days	Head position	Occurrence of stereotype	Faeces color			Urine color
		Normal	None	Normal	Pale	Dark	Normal	Pale/dark yellow
1,2,3	1st Day	100(3)	100(3)	100(3)	Nil	Nil	100(3)	Nil
	8th Day	100(3)	100(3)	100(3)	Nil	Nil	100(3)	Nil
	15th Day	100(3)	100(3)	100(3)	Nil	Nil	100(3)	Nil
4,5	1st Day	100(2)	100(2)	100(2)	Nil	Nil	100(2)	Nil
	8th Day	100(2)	100(2)	100(2)	Nil	Nil	100(2)	Nil
	15th Day	100(2)	100(2)	100(2)	Nil	Nil	100(2)	Nil
6,7,8	1st Day	100(3)	100(3)	100(3)	Nil	Nil	100(3)	Nil
	8th Day	100(3)	100(3)	100(3)	Nil	Nil	100(3)	Nil
	15th Day	100(3)	100(3)	100(3)	Nil	Nil	100(3)	Nil
9,10	1st Day	100(2)	100(2)	100(2)	Nil	Nil	100(2)	Nil
	8th Day	100(2)	100(2)	100(2)	Nil	Nil	100(2)	Nil
	15th Day	100(2)	100(2)	100(2)	Nil	Nil	100(2)	Nil

Table – 6 SUB ACUTE TOXICITY STUDYPRE TERMINAL DEATH(S) TABLE

S.No	Days of observation	VC		LD		MD		HD
		M	F	M	F	M	F	M	F
1	1	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
2	2	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
3	3	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
4	4	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
5	5	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
6	6	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
7	7	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
8	8	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
9	9	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
10	10	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
11	11	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
12	12	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
13	13	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
14	14	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
15	15	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
16	16	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
17	17	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
18	18	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
19	19	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
20	20	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
21	21	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
22	22	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
23	23	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
24	24	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
25	25	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
26	26	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
27	27	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6
28	28	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6

Table – 7 Home cage observations

Name of the Test item: Milnacipran hydrochloride

Groups	Days	Body posture (%)		Respiration character (%)		Tremors
		Normal	Abnormal	Normal	Abnormal	None
VC	1st Day	100(12)	Nil	100(12)	Nil	100(12)
	8th Day	100(12)	Nil	100(12)	Nil	100(12)
	15th Day	100(12)	Nil	100(12)	Nil	100(12)
	22ndDay	100(12)	Nil	100(12)	Nil	100(12)
	28th Day	100(12)	Nil	100(12)	Nil	100(12)
LD	1st Day	100(12)	Nil	100(12)	Nil	100(12)
	8th Day	100(12)	Nil	100(12)	Nil	100(12)
	15th Day	100(12)	Nil	100(12)	Nil	100(12)
	22ndDay	100(12)	Nil	100(12)	Nil	100(12)
	28th Day	100(12)	Nil	100(12)	Nil	100(12)
MD	1st Day	100(12)	Nil	100(12)	Nil	100(12)
	8th Day	100(12)	Nil	100(12)	Nil	100(12)
	15th Day	100(12)	Nil	100(12)	Nil	100(12)
	22ndDay	100(12)	Nil	100(12)	Nil	100(12)
	28th Day	100(12)	Nil	100(12)	Nil	100(12)
HD	1st Day	100(12)	Nil	100(12)	Nil	100(12)
	8th Day	100(12)	Nil	100(12)	Nil	100(12)
	15th Day	100(12)	Nil	100(12)	Nil	100(12)
	22ndDay	100(12)	Nil	100(12)	Nil	100(12)
	28th Day	100(12)	Nil	100(12)	Nil	100(12)

Table – 8: Home cage observations (contd.)

Name of the Test item: Milnacipran hydrochloride

Groups	Days	Convulsions	Vocalization	Palpebral closure
		None		Open	Eyelids slightly lowered
VC	1st Day	100(12)	Nil	100(12)	Nil
	8th Day	100(12)	Nil	100(12)	Nil
	15th Day	100(12)	Nil	100(12)	Nil
	22ndDay	100(12)	Nil	100(12)	Nil
	28th Day	100(12)	Nil	100(12)	Nil
LD	1st Day	100(12)	Nil	100(12)	Nil
	8th Day	100(12)	Nil	100(12)	Nil
	15th Day	100(12)	Nil	100(12)	Nil
	22ndDay	100(12)	Nil	100(12)	Nil
	28th Day	100(12)	Nil	100(12)	Nil
MD	1st Day	100(12)	Nil	100(12)	Nil
	8th Day	100(12)	Nil	100(12)	Nil
	15th Day	100(12)	Nil	100(12)	Nil
	22ndDay	100(12)	Nil	100(12)	Nil
	28th Day	100(12)	Nil	100(12)	Nil
HD	1st Day	100(12)	Nil	100(12)	Nil
	8th Day	100(12)	Nil	100(12)	Nil
	15th Day	100(12)	Nil	100(12)	Nil
	22ndDay	100(12)	Nil	100(12)	Nil
	28h Day	100(12)	Nil	100(12)	Nil

Table – 9 Open field activity

Name of the Test item: Milnacipran hydrochloride

Groups	Days	No. of rearings	Gait	Tail elevation
		Normal	Normal	Lifted while walking	Occasionally lifted
VC	1st Day	100(12)	100(12)	100(12)	Nil
	8th Day	100(12)	100(12)	100(12)	Nil
	15th Day	100(12)	100(12)	100(12)	Nil
	22ndDay	100(12)	100(12)	100(12)	Nil
	28th Day	100(12)	100(12)	100(12)	Nil
LD	1st Day	100(12)	100(12)	100(12)	Nil
	8th Day	100(12)	100(12)	100(12)	Nil
	15th Day	100(12)	100(12)	100(12)	Nil
	22ndDay	100(12)	100(12)	100(12)	Nil
	28th Day	100(12)	100(12)	100(12)	Nil
MD	1st Day	100(12)	100(12)	100(12)	Nil
	8th Day	100(12)	100(12)	100(12)	Nil
	15th Day	100(12)	100(12)	100(12)	Nil
	22ndDay	100(12)	100(12)	100(12)	Nil
	28th Day	100(12)	100(12)	100(12)	Nil
HD	1st Day	100(12)	100(12)	100(12)	Nil
	8th Day	100(12)	100(12)	100(12)	Nil
	15th Day	100(12)	100(12)	100(12)	Nil
	22ndDay	100(12)	100(12)	100(12)	Nil
	28th Day	100(12)	100(12)	100(12)	Nil

Table – 10 Open field activity (Contd.)

Name of the Test item: Milnacipran hydrochloride

Groups	Days	Head position	Occurrence of stereotype	Faeces color			Urine color
		Normal	None	Normal	Pale	Dark	Normal	Pale/dark yellow
VC	1st Day	100(12)	100(12)	100(12)	Nil	Nil	100(12)	Nil
	8th Day	100(12)	100(12)	100(12)	Nil	Nil	100(12)	Nil
	15th Day	100(12)	100(12)	100(12)	Nil	Nil	100(12)	Nil
	22ndDay	100(12)	100(12)	100(12)	Nil	Nil	100(12)	Nil
	28th Day	100(12)	100(12)	100(12)	Nil	Nil	100(12)	Nil
LD	1st Day	100(12)	100(12)	100(12)	Nil	Nil	100(12)	Nil
	8th Day	100(12)	100(12)	100(12)	Nil	Nil	100(12)	Nil
	15th Day	100(12)	100(12)	100(12)	Nil	Nil	100(12)	Nil
	22ndDay	100(12)	100(12)	100(12)	Nil	Nil	100(12)	Nil
	28th Day	100(12)	100(12)	100(12)	Nil	Nil	100(12)	Nil
MD	1st Day	100(12)	100(12)	100(12)	Nil	Nil	100(12)	Nil
	8th Day	100(12)	100(12)	100(12)	Nil	Nil	100(12)	Nil
	15th Day	100(12)	100(12)	100(12)	Nil	Nil	100(12)	Nil
	22ndDay	100(12)	100(12)	100(12)	Nil	Nil	100(12)	Nil
	28th Day	100(12)	100(12)	100(12)	Nil	Nil	100(12)	Nil
HD	1st Day	100(12)	100(12)	100(12)	Nil	Nil	100(12)	Nil
	8th Day	100(12)	100(12)	100(12)	Nil	Nil	100(12)	Nil
	15th Day	100(12)	100(11)	100(12)	Nil	Nil	100(12)	Nil
	22ndDay	100(12)	100(11)	100(12)	Nil	Nil	100(12)	Nil
	28th Day	100(12)	100(10)	100(12)	Nil	Nil	100(12)	Nil

Table – 11 Histopathological observations

Organ wise lesions	Vehicle control			High dose
	M	F	M		F
Kidney
1.Congestion and haemorrhage	0/6	0/6	0/6		0/6
2.Tubular degeneration	0/6	2/6	6/6		6/6
3.Inflammatory Foci and infilteration of inflammatory cells	6/6	6/6	6/6		6/6
Liver
1.Congestion and haemorrhage	4/6	0/6	0/6		0/6
2. Degeneration and necrosis	6/6	6/6	6/6		6/6
3.Inflammation and infiltration of inflammatory cells	6/6	6/6	6/6		6/6
Lungs
1.Congestion and haemorrhage	0/6	0/6	0/6		0/6
2.Inflammatory foci with infiltration of inflammatory cells	6/6	6/6	6/6		6/6
Heart
1.Congestion and haemorrhage	2/6	0/6	2/6		0/6
2.Degeneration and necrosis	0/6	4/6	2/6		0/6
3.Inflammatory foci and infiltration of inflammatory cells	2/6	2/6	4/6		0/6
4.Pericarditis/ myocarditis	1/6	0/6	0/6		2/6
Stomach and Intestines
1.Inflammation	0/6	0/6	0/6		1/6
2.Sub mucosal lymphocytic infiltration	0/6	0/6	0/6		0/6
3.Cystic degeneration	1/6	1/6	0/6		0/6
Spleen
1.Congestion and haemorrhage	0/6	0/6	0/6		0/6
2.Lymphoid follicular depletion	0/6	0/6	0/6		0/6
Brain
1.Meningeal congestion and haemorrhage	2/6	0/6	0/6		0/6
2.Epithelial cell hyperplasia	0/6	0/6	0/6		0/6
3.Foci of inflammation	1/6	0/6	0/6		0/6
4.Demyelination/degeneration	2/6	0/6	0/6		0/6
Testes
1. Seminiferous tubular degeneration	0/6	-	0/6		-

CONCLUSION:

In the present study, no mortality was found in any group of animals after single oral administration of Milnacipran hydrochloride in wistar rats. No signs and symptoms of toxicity were observed in all the treated groups. All the animals appeared to be normal during the study period. There were no deaths related to the toxicity of the test article received up to a maximum dose of 45 mg/kg once during the study period. The gross pathology revealed no abnormalities in the appearance of major vital organs related to test item. The food intake of the animals in treated groups was observed to be normal in all treated groups. The body weight in treated groups was normal. The results of repeated dose 28 days toxicity study have no lethality observed in treated groups animals. There were no significant changes recorded in physical, physiological, hematological and biochemical parameters under experimental conditions in Wistar rats. All the results of acute and sub acute toxicity data were given in Table – 2 to 11.

The histopathology observations give us information that acceptable toxicity levels are present such as gastric ulcers and lungs. The food intake of the animals in treated groups was observed to be normal in all treated groups. The body weight in treated groups was normal. Few animals showed mild inflammatory changes with infiltration of inflammatory cells in myocardium of vehicle control and high dose group animals.

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Received on 30.10.2012

Modified on 02.12.2012

Accepted on 18.12.2012

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Research J. Pharmacology and Pharmacodynamics. 5(1): January –February 2013, 51-57