Anti-diabetic Drugs की Pharmacological एवं Toxicological Screening

परिभाषा (Definition)

1. उद्देश्य (Objectives)

1. Test drug की blood glucose कम करने की क्षमता जानना।
2. Test drug की तुलना standard drug से करना।
3. Drug की dose, efficacy और duration of action पता करना।
4. Drug की toxicity / safety जाँचना।
5. यह देखना कि drug insulin secretion बढ़ाती है, insulin sensitivity बढ़ाती है या glucose absorption कम करती है।

2. सामान्य आवश्यकताएँ (Requirements)

• Healthy albino rats / mice
• Glucometer या blood glucose estimation kit
• Test drug / plant extract
• Standard drug: Glibenclamide या Metformin
• Alloxan monohydrate या Streptozotocin (STZ)
• Glucose solution
• Normal saline / distilled water
• Oral feeding cannula, syringe, weighing balance
• Animal ethical committee approval

Note: Animal experimentation ethical approval तथा institutional rules के अनुसार ही किया जाता है।

3. Pharmacological Screening of Anti-diabetic Drugs

1. Oral Glucose Tolerance Test (OGTT)
2. Alloxan-induced diabetic model
3. Streptozotocin-induced diabetic model

A. Oral Glucose Tolerance Test (OGTT)

Principle

Procedure

Flow chart

Healthy rats/mice select करो
          ↓
Overnight fasting कराओ (लगभग 12 घंटे)
          ↓
Animals को अलग-अलग groups में बाँटो
          ↓
Control, Standard और Test drug दो
          ↓
30 min बाद glucose solution orally दो
          ↓
Blood sample लो
(0, 30, 60, 120 min पर)
          ↓
Blood glucose level measure करो
          ↓
Groups के results compare करो

Grouping

Detailed Procedure

1. Rats/mice को लगभग 12 घंटे fast कराया जाता है। Water दिया जा सकता है।
2. Initial fasting blood glucose level note किया जाता है।
3. Animals को 4-5 groups में divide किया जाता है।
4. Control group को vehicle दिया जाता है।
5. Standard group को glibenclamide या metformin दिया जाता है।
6. Test groups को अलग-अलग dose में test drug दिया जाता है।
7. लगभग 30 मिनट बाद सभी relevant groups को glucose solution orally दिया जाता है।
8. Blood sample tail vein से लिया जाता है।
9. Blood glucose level 0, 30, 60 और 120 minutes पर estimate किया जाता है।
10. जिस group में glucose level कम बढ़े या जल्दी normal हो जाए, उसमें anti-diabetic activity मानी जाती है।

Observation / Result

• Glucose control group में blood glucose level अधिक बढ़ता है।
• Standard group में glucose level control group से कम होता है।
• यदि test drug group में blood glucose significantly कम होता है, तो test drug में anti-diabetic / anti-hyperglycemic activity होती है।

Percentage Reduction Formula

Advantages

• Simple method
• कम समय में result मिलता है
• Normal animals में भी किया जा सकता है
• Glucose tolerance पर drug का effect पता चलता है

Limitation

• यह chronic diabetes model नहीं है।
• यह pancreatic beta-cell damage वाले diabetic animals को fully represent नहीं करता।

B. Alloxan-Induced Diabetic Model

Principle

Procedure

Flow chart

Animals को overnight fast कराओ
          ↓
Initial blood glucose measure करो
          ↓
Alloxan inject करके diabetes induce करो
          ↓
कुछ समय बाद blood glucose check करो
          ↓
Diabetic animals select करो
          ↓
Groups बनाओ
          ↓
Control / Standard / Test drug treatment दो
          ↓
निर्धारित दिनों तक blood glucose monitor करो
          ↓
Results compare करके anti-diabetic activity calculate करो

Detailed Procedure

1. Healthy albino rats/mice को select किया जाता है।
2. Animals को लगभग 12 घंटे fast कराया जाता है।
3. Baseline fasting blood glucose level measure किया जाता है।
4. Alloxan solution तैयार करके animals को inject किया जाता है, जिससे diabetes induce होती है।
5. Alloxan देने के बाद animals को glucose solution दिया जा सकता है ताकि sudden hypoglycemia से बचाया जा सके।
6. लगभग 48-72 घंटे बाद blood glucose level check किया जाता है।
7. जिन animals में blood glucose level काफी अधिक हो जाता है, उन्हें diabetic animals माना जाता है।
8. Diabetic animals को groups में divide किया जाता है:

1. Test drug और standard drug को daily, usually oral route से, निर्धारित अवधि तक दिया जाता है।
2. Fasting blood glucose level को Day 0, Day 7, Day 14 आदि पर estimate किया जाता है।
3. Body weight, food intake, water intake और urine output भी note किए जा सकते हैं।
4. अंत में groups के blood glucose levels compare किए जाते हैं।

Observation

• Normal control: Blood glucose normal रहता है।
• Diabetic control: Blood glucose बहुत बढ़ा हुआ रहता है।
• Standard group: Blood glucose में marked reduction होता है।
• Test group: यदि glucose level diabetic control से significantly कम हो, तो test drug anti-diabetic activity दिखाती है।

Result

Merits

• Diabetes का reliable experimental model है।
• Insulin deficiency वाली diabetes में useful है।
• कई दिनों तक treatment effect देखा जा सकता है।

Demerits

• Alloxan beta cells को damage करता है, इसलिए severe diabetes हो सकती है।
• Dose variation से animal mortality हो सकती है।
• Proper handling और ethical precautions जरूरी हैं।

C. Streptozotocin (STZ)-Induced Diabetic Model

Principle

Short Procedure

Fasting animals
      ↓
STZ administration
      ↓
48-72 hours बाद blood glucose estimation
      ↓
Diabetic animals select करना
      ↓
Test drug और standard drug देना
      ↓
Blood glucose और body weight record करना
      ↓
Anti-diabetic effect evaluate करना

4. Standard Drugs Used

5. Toxicological Screening of Anti-diabetic Drug

Definition

Objectives

1. Safe dose पता करना
2. Toxic dose पता करना
3. Mortality देखना
4. Side effects जैसे sedation, tremors, diarrhea, convulsions आदि observe करना
5. आगे pharmacological study के लिए proper dose select करना

Acute Oral Toxicity Study

Principle

Procedure Chart

Healthy animals select करो
          ↓
Overnight fasting
          ↓
Test drug की different doses orally दो
          ↓
पहले 4 घंटे continuously observe करो
          ↓
24 घंटे तक mortality देखो
          ↓
14 days तक animals observe करो
          ↓
Safe dose और toxic dose determine करो

Detailed Procedure

1. Healthy animals select किए जाते हैं।
2. Animals को overnight fast कराया जाता है।
3. Test drug को oral route से दिया जाता है।
4. Animals को पहले 30 minutes, 1 hour, 2 hours, 4 hours और फिर 24 hours तक observe किया जाता है।
5. आगे लगभग 14 days तक observation किया जाता है।
6. निम्न signs देखे जाते हैं:
   • Mortality
   • Tremors
   • Convulsions
   • Sedation
   • Excess salivation
   • Diarrhea
   • Change in skin/fur
   • Change in food and water intake
   • Change in body weight
   • Hypoglycemia signs जैसे weakness, sweating-like response, reduced movement या coma
7. जिस dose पर toxicity न हो, उसे safe dose माना जाता है।
8. Pharmacological screening के लिए अक्सर safe dose का एक fraction, जैसे 1/10th या 1/5th, चुना जाता है।

Toxicity study current institutional animal ethics and OECD-based protocols के अनुसार qualified personnel द्वारा ही की जानी चाहिए।

6. Precautions

1. सभी animals को समान conditions में रखें।
2. Proper fasting कराएँ, लेकिन पानी उपलब्ध रखें।
3. Alloxan/STZ freshly prepare करें क्योंकि ये unstable हो सकते हैं।
4. Blood sampling का time सभी groups में same रखें।
5. Control, standard और test groups अवश्य रखें।
6. Animals को random तरीके से groups में divide करें।
7. Glucose measurement calibrated glucometer/validated method से करें।
8. Data को mean ± SEM के रूप में present करें और statistical analysis करें।
9. Animal ethics approval और humane handling अनिवार्य है।

निष्कर्ष (Conclusion)

Exam में लिखने के लिए एक लाइन

Pharmacological and Toxicological Screening of Anti-diabetic Drugs

Definition

Objectives

1. To find out the blood glucose lowering effect of the test drug.
2. To compare the test drug with a standard anti-diabetic drug.
3. To determine the safe and effective dose of the test drug.
4. To study toxic effects and mortality produced by the drug.

A. Pharmacological Screening

1. Oral Glucose Tolerance Test (OGTT)
2. Alloxan-induced diabetic model
3. Streptozotocin-induced diabetic model

1. Oral Glucose Tolerance Test (OGTT)

Definition

Principle

Requirements

• Healthy albino rats or mice
• Test drug or plant extract
• Standard drug: Glibenclamide or Metformin
• Glucose solution
• Normal saline or vehicle
• Glucometer or blood glucose estimation kit
• Oral feeding needle and syringe

Grouping of Animals

Procedure

1. Select healthy albino rats or mice.
2. Keep animals fasting overnight, usually for 12 hours. Water is allowed.
3. Measure the fasting blood glucose level.
4. Divide animals into different groups.
5. Give vehicle to the control group.
6. Give standard drug to the standard group.
7. Give test drug in selected doses to the test groups.
8. After 30 minutes, give glucose solution orally to all groups except normal control.
9. Collect blood from tail vein at 0, 30, 60 and 120 minutes.
10. Estimate blood glucose level by glucometer or biochemical method.
11. Compare blood glucose levels of test group with glucose control group.

Flow Chart

Healthy animals
       ↓
Overnight fasting
       ↓
Measure fasting blood glucose
       ↓
Divide into groups
       ↓
Give vehicle / standard drug / test drug
       ↓
After 30 minutes give oral glucose
       ↓
Measure blood glucose at 0, 30, 60 and 120 minutes
       ↓
Compare results

Observation

• In glucose control group, blood glucose level increases markedly.
• In standard drug group, blood glucose level decreases.
• If test drug reduces the increased blood glucose level, it shows anti-diabetic activity.

Result

Advantages

• Simple and quick method.
• Useful for screening anti-hyperglycemic drugs.
• Can be performed in normal animals.

Limitation

2. Alloxan-Induced Diabetic Model

Definition

Principle

Requirements

• Albino rats or mice
• Alloxan monohydrate
• Test drug
• Standard drug: Glibenclamide or Metformin
• Glucometer or blood glucose estimation kit
• Normal saline or vehicle
• Oral feeding needle and syringe

Grouping

Procedure

1. Select healthy albino rats or mice.
2. Fast the animals overnight.
3. Record initial fasting blood glucose level.
4. Administer alloxan to induce diabetes.
5. After alloxan administration, animals may be given glucose solution to prevent sudden hypoglycemia.
6. After 48 to 72 hours, measure blood glucose level.
7. Select animals showing increased blood glucose level as diabetic animals.
8. Divide diabetic animals into different groups.
9. Give test drug to test groups and standard drug to standard group daily for a fixed period.
10. Measure fasting blood glucose level at regular intervals, such as Day 0, Day 7 and Day 14.
11. Also record body weight, food intake and water intake if required.
12. Compare the blood glucose levels of all groups.

Flow Chart

Healthy animals
       ↓
Overnight fasting
       ↓
Record fasting blood glucose
       ↓
Administer alloxan
       ↓
Wait for 48-72 hours
       ↓
Check blood glucose and select diabetic animals
       ↓
Divide into control, standard and test groups
       ↓
Give treatment for selected period
       ↓
Measure blood glucose regularly
       ↓
Compare results

Observation

• Normal control group shows normal blood glucose level.
• Diabetic control group shows high blood glucose level.
• Standard drug group shows reduction in blood glucose level.
• Test drug group shows reduction in blood glucose level if the drug is active.

Result

Advantages

• Commonly used and reliable method.
• Useful for studying drugs in diabetic animals.
• Effect of repeated treatment can be studied.

Disadvantages

• Alloxan may produce severe diabetes and animal mortality.
• Proper handling is required.
• It mainly represents insulin-deficient diabetes.

3. Streptozotocin-Induced Diabetic Model

Principle

Procedure

Fast animals
       ↓
Administer streptozotocin
       ↓
After 48-72 hours measure blood glucose
       ↓
Select diabetic animals
       ↓
Give standard drug and test drug
       ↓
Measure blood glucose at regular intervals
       ↓
Compare anti-diabetic activity

Result

B. Toxicological Screening

Definition

Objective

1. To determine the safety of the test drug.
2. To identify toxic effects.
3. To

Pharmacological and Toxicological Screening of Anti-diabetic Drugs

Definition

Objectives

1. To evaluate the blood glucose lowering effect of the test drug.
2. To compare the test drug with a standard anti-diabetic drug.
3. To find the effective dose of the test drug.
4. To study the toxicity and safety of the test drug.

A. Pharmacological Screening of Anti-diabetic Drugs

1. Oral Glucose Tolerance Test (OGTT)
2. Alloxan-induced diabetic model
3. Streptozotocin-induced diabetic model

1. Oral Glucose Tolerance Test (OGTT)

Principle

Animals Required

Drugs Required

• Test drug or plant extract
• Standard drug: Glibenclamide or Metformin
• Glucose solution
• Normal saline or vehicle

Grouping of Animals

Procedure

1. Select healthy albino rats or mice.
2. Keep the animals fasting overnight for about 12 hours. Water is allowed.
3. Measure fasting blood glucose level.
4. Divide the animals into different groups.
5. Give vehicle to the normal control group.
6. Give standard drug to the standard group.
7. Give the test drug to the test groups.
8. After 30 minutes, administer glucose solution orally.
9. Collect blood samples from the tail vein at 0, 30, 60 and 120 minutes.
10. Measure blood glucose level by glucometer or biochemical method.
11. Compare the blood glucose level of test groups with the glucose control group.

Flow Chart

Healthy animals
        ↓
Overnight fasting
        ↓
Measure fasting blood glucose
        ↓
Divide animals into groups
        ↓
Give vehicle / standard drug / test drug
        ↓
After 30 minutes give oral glucose
        ↓
Measure blood glucose at 0, 30, 60 and 120 minutes
        ↓
Compare the results

Observation

• Blood glucose level rises markedly in the glucose control group.
• Standard drug reduces the blood glucose level.
• If the test drug reduces the increased blood glucose level, it shows anti-diabetic activity.

Result

Advantages

• It is simple and rapid.
• It is useful for preliminary screening.
• It can be performed in normal animals.

Limitation

2. Alloxan-Induced Diabetic Model

Principle

Drugs Required

• Alloxan monohydrate
• Test drug
• Standard drug: Glibenclamide or Metformin
• Normal saline or vehicle
• Glucose solution
• Glucometer or blood glucose estimation kit

Grouping of Animals

Procedure

1. Select healthy albino rats or mice.
2. Fast the animals overnight.
3. Record fasting blood glucose level.
4. Administer alloxan to induce diabetes.
5. After alloxan administration, give glucose solution to prevent sudden hypoglycemia.
6. After 48 to 72 hours, estimate blood glucose level.
7. Animals with high blood glucose level are selected as diabetic animals.
8. Divide diabetic animals into different groups.
9. Give standard drug to the standard group and test drug to the test groups daily for a fixed period.
10. Measure fasting blood glucose level on Day 0, Day 7 and Day 14.
11. Body weight, food intake and water intake may also be recorded.
12. Compare the results with the diabetic control group.

Flow Chart

Healthy animals
        ↓
Overnight fasting
        ↓
Record fasting blood glucose
        ↓
Administer alloxan
        ↓
After 48-72 hours check blood glucose
        ↓
Select diabetic animals
        ↓
Divide into groups
        ↓
Give standard drug and test drug
        ↓
Measure blood glucose regularly
        ↓
Compare results

Observation

• Normal control group shows normal blood glucose level.
• Diabetic control group shows high blood glucose level.
• Standard drug group shows a marked decrease in blood glucose level.
• Test drug group shows reduction in blood glucose level if it has anti-diabetic activity.

Result

Advantages

1. It is a commonly used model.
2. It is useful for studying drugs in diabetic animals.
3. Repeated drug treatment can be studied.

Disadvantages

1. Alloxan may produce severe diabetes.
2. It may cause animal mortality.
3. Proper handling is necessary.

3. Streptozotocin-Induced Diabetic Model

Principle

Procedure

1. Fast the animals overnight.
2. Record fasting blood glucose level.
3. Administer streptozotocin to the animals.
4. After 48 to 72 hours, measure blood glucose level.
5. Select animals with high blood glucose level as diabetic animals.
6. Divide animals into diabetic control, standard and test groups.
7. Give the standard drug and test drug for a fixed period.
8. Measure blood glucose level at regular intervals.
9. Compare the results with diabetic control group.

Result

B. Toxicological Screening of Anti-diabetic Drugs

Definition

Objectives

1. To determine the safe dose of the test drug.
2. To determine toxic signs produced by the drug.
3. To observe mortality, if any.
4. To select a safe dose for pharmacological screening.

Acute Oral Toxicity Study

Principle

Procedure

1. Select healthy animals.
2. Keep the animals fasting overnight.
3. Divide animals into suitable groups.
4. Administer the test drug orally in different doses.
5. Observe animals continuously for the first 4 hours.
6. Record mortality after 24 hours.
7. Continue observation for 14 days.
8. Record changes in body weight, food intake and water intake.
9. Select a safe dose for further pharmacological study.

Flow Chart

Healthy animals
        ↓
Overnight fasting
        ↓
Give test drug orally in selected doses
        ↓
Observe animals for 4 hours
        ↓
Record mortality after 24 hours
        ↓
Observe for 14 days
        ↓
Determine safe dose and toxic dose

Toxic Signs Observed

• Decreased activity
• Sedation
• Tremors
• Convulsions
• Salivation
• Diarrhoea
• Change in food intake
• Change in water intake
• Change in body weight
• Weakness
• Death or mortality

Result

Precautions

1. Use healthy animals of similar weight.
2. Keep all animals under the same laboratory conditions.
3. Fast animals properly before blood glucose estimation.
4. Use control, standard and test groups in every experiment.
5. Measure blood glucose at the same time in all groups.
6. Handle alloxan and streptozotocin carefully.
7. Record all observations properly.
8. Perform experiments only after approval of the institutional animal ethics committee.

Conclusion

“The test drug is considered to possess anti-diabetic activity if it produces a significant reduction in blood glucose level when compared with the diabetic control group and shows an effect comparable to the standard drug.”