Diabetes Mellitus (DM)

Diabetes Mellitus (DM) is a group of metabolic disorders characterized by high blood glucose levels
(hyperglycemia) due to insufficient insulin production, insulin resistance, or both. It can lead to serious
complications if not managed properly.
Type of Diabetes Description Key Features
Mellitus
Type 1 Diabetes Autoimmune destruction of - Typically diagnosed in children/adolescents
insulin-producing beta cells. - Requires lifelong insulin therapy
- Rapid onset of symptoms
Type 2 Diabetes Insulin resistance and relative - Most common type
insulin deficiency. - Often linked to obesity and sedentary lifestyle
- Managed with lifestyle changes and
medications
Gestational Diabetes Diabetes that develops during - Usually resolves after childbirth
pregnancy. - Increases risk of Type 2 diabetes later

Oral Hypoglycemic agent

Oral hypoglycemics, also known as oral antidiabetic agents, are medications taken by mouth to help
lower blood glucose levels in individuals with diabetes, particularly Type 2 diabetes. They work through
various mechanisms to improve insulin sensitivity, increase insulin secretion, decrease glucose
production by the liver, or slow carbohydrate absorption from the digestive tract.
Classification
Class Examples Mechanism of Action (MOA)
1. Biguanide - Metformin Decreases hepatic glucose production and increases insulin
sensitivity.
2. Insulin Sulfonylureas
Secretagogues
- Tolbutamide KATP channel blockers, stimulating insulin release from the
pancreas.
- Glibenclamide,
Glipizide, Gliclazide,
Glimepiride
Meglitinides
- Repaglinide, Stimulate rapid insulin secretion from the pancreas.
Nateglinide
3. - Troglitazone, Improve insulin sensitivity in muscle and adipose tissue.
Thiazolidinediones Pioglitazone
4. GLP-1 - Exenatide, Liraglutide, Enhance glucose-dependent insulin secretion and slow gastric
Analogues Albiglutide, Dulaglutide emptying.
5. DPP-4 Inhibitors - Sitagliptin, Vildagliptin, Inhibit DPP-4 enzyme, increasing incretin levels, leading to
Saxagliptin, Linagliptin, more insulin secretion and reduced glucagon.
Alogliptin
6. α-Glucosidase - Acarbose, Miglitol, Delay carbohydrate absorption in the intestines, reducing
Inhibitors Voglibose postprandial glucose levels.
7. Amylin Analog - Pramlintide Slows gastric emptying, promotes satiety, and reduces
postprandial glucagon secretion.
8. SGLT-2 - Dapagliflozin, Promote renal excretion of glucose, reducing blood glucose
Inhibitors Remogliflozin levels.
1. Insulin Secretagogues
A. Sulfonylureas
Sulfonylureas are a class of medications derived from sulfonamides, initially noted for their antibacterial
effects. The observation that these compounds caused hypoglycemia in typhoid patients led to the
development of sulfonylureas, which are designed to enhance insulin secretion from the pancreas.
Mechanism of Action
Binding Mechanism of Sulfonylureas
 Sulfonylureas bind to sulfonylurea receptors (SUR), which are ATP-sensitive potassium channels
(KATP) located on the membrane of pancreatic β-cells.
 By binding to the SUR1 subunit, sulfonylureas close these potassium channels, preventing
potassium efflux. This closure causes depolarization of the membrane, which opens voltage-
dependent calcium channels, resulting in calcium influx.
 The increased calcium levels trigger the release of insulin stored in the granules of the β-cells.

Sulfonylureas lower blood glucose levels through several mechanisms:

1. Insulin Secretion: Stimulate the release of insulin from pancreatic β-cells.
2. Insulin Sensitivity: Increase the sensitivity of peripheral tissues to insulin.
3. Insulin Receptors: Enhance the number of insulin receptors on target tissues.
4. Hepatic Gluconeogenesis: Suppress hepatic gluconeogenesis, reducing glucose production by
the liver.
5. Glucagon Levels: Prolonged use in patients with Type 2 Diabetes Mellitus (DM) can lead to
reduced glucagon levels, likely due to negative feedback from elevated insulin levels.
Pharmacokinetics
 Absorption: Well-absorbed orally.
 Protein Binding: Highly bound to plasma proteins (>90%).
 Metabolism: Primarily metabolized in the liver; some metabolites excreted in urine.
 Caution: Avoid in patients with renal or hepatic dysfunction.
Adverse Effects
 Common: Hypoglycemia (lower risk with tolbutamide).
 Others: Nausea, vomiting, jaundice, allergic reactions.
 Alcohol Interaction: Can cause a disulfiram-like reaction; patients should avoid alcohol.
Drug Interactions with Sulfonylureas
I. Augment Hypoglycemic Effect
 Displacing Agents: NSAIDs, warfarin, sulfonamides (reduce protein binding).
 Metabolism Inhibitors: Alcohol, chloramphenicol, cimetidine (increase levels).
II. Reduce Effects
 Counteracting Agents: Diuretics and glucocorticoids (increase blood glucose).

Second Generation Agents

Overview: Second generation sulfonylureas are more potent than first generation agents, with fewer side
effects and drug interactions. However, they can still cause hypoglycemia, necessitating cautious use,
especially in the elderly and those with cardiovascular diseases. These agents are contraindicated in
patients with renal or hepatic impairment.
Glibenclamide (Gliburide):
 Characteristics: Longer-acting; can be administered once daily.
 Dosage: Start with 2.5 mg in the morning, with potential increases to 5–10 mg once daily.
 Side Effects: Can cause hypoglycemia; may also induce flushing after alcohol consumption.
 Formulations: Available in controlled release and extended release options.
Second Generation Agents
Glipizide:
 Characteristics: Short half-life; food delays absorption, so take 30 minutes before breakfast.
 Hypoglycemia Risk: Less likely due to short half-life.
 Dosage: Start at 5 mg/day, may increase to 15 mg/day.
 Metabolism: Liver; contraindicated in renal and hepatic dysfunction.
 Brand Names: GLYNASE, GLIBETIC, D-GLIP (5 mg tablet).
Gliclazide:
 Use: Suitable for patients with renal dysfunction; may delay onset of retinopathy.
 Dosage: 40–240 mg once daily.
 Brand Names: D-GLIC, GLIX.
Glimepiride:
 Characteristics: Longer-acting; can be taken as a single morning dose.
 Dosage: Start at 1 mg, may increase to 4 mg daily (maximum 8 mg).
 Brand Names: GLYPRIDE, GLIMZ, AMARYL (1, 2 mg tablets).
B. Meglitinides
Overview: Repaglinide and nateglinide are insulin secretagogues that enhance insulin release by
blocking ATP-dependent potassium channels (sulfonylurea receptors) in pancreatic β-cells.
This summary presents the key information about glipizide, gliclazide, glimepiride, and the meglitinides
clearly and concisely.
Glucagon-Like Peptide-1 (GLP-1) Analogs/Incretin Analogs
Overview: GLP-1, an incretin hormone released from the gut after oral glucose intake, enhances insulin
secretion. However, it cannot be used therapeutically due to rapid degradation by dipeptidyl peptidase-4
(DPP-4).
Mechanism of Action (MOA)
GLP-1 analogs act on GLP-1 receptors to:
 Enhance Insulin Secretion: Increase insulin release from pancreatic β-cells in response to
elevated glucose levels.
 Suppress Glucagon Release: Decrease glucagon secretion, reducing hepatic glucose production.
 Delay Gastric Emptying: Slow the rate at which food leaves the stomach, leading to a more
gradual absorption of glucose.
 Reduce Appetite: Promote satiety, aiding in weight management.
Agents
Exenatide:
 Type: Synthetic GLP-1 analog.
 Dosage: 5–10 µg subcutaneously 30–60 minutes before meals, twice daily. Long-acting forms are
available for weekly injection.
 Effects: Increases β-cell mass and lowers HbA1c levels.
Liraglutide:
 Type: Longer-acting GLP-1 analog.
 Dosage: Start at 0.6 mg once daily; can be doubled after one week. Often used as an add-on
therapy.
Albiglutide and Dulaglutide:
 Characteristics: Fused to human albumin, allowing for longer half-lives suitable for weekly
injections.
Other GLP-1 Analogs: Semaglutide and lixisenatide.
Adverse Effects
 Common: Nausea, vomiting (self-limiting), diarrhea, weight loss.
 Serious but Rare: Hemorrhagic pancreatitis, which can be fatal.
 Caution: Should be avoided in patients with renal impairment, as it may exacerbate conditions.

Dipeptidyl Peptidase-4 (DPP-4) Inhibitors

Dipeptidyl peptidase-4 (DPP-4) is an enzyme that degrades incretins, hormones that stimulate insulin
secretion in response to meals.
Mechanism of Action (MOA) of DPP-4 Inhibitors
DPP-4 inhibitors, including sitagliptin, enhance the action of incretin hormones by preventing their
degradation. Here’s a more detailed breakdown of their mechanism:
1.Inhibition of DPP-4 Enzyme:
Role of DPP-4: Dipeptidyl peptidase-4 (DPP-4) is an enzyme that rapidly inactivates incretins, primarily
glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP).
Effect of Inhibition: By inhibiting DPP-4, these medications increase the half-life and overall
concentration of active incretins in the bloodstream, allowing them to exert their physiological effects for
a longer duration.
2. Enhancement of Insulin Secretion:
Mechanism: Elevated levels of GLP-1 and GIP stimulate insulin secretion from the pancreatic β-cells in a
glucose-dependent manner. This means that insulin is released primarily when blood glucose levels are
elevated, reducing the risk of hypoglycemia.
Signal Transduction: When incretins bind to their receptors on β-cells, they activate signaling pathways
(such as the cAMP pathway), leading to increased calcium influx and ultimately promoting insulin
granule exocytosis.
3. Decrease in Glucagon Levels:
Mechanism: Incretins inhibit glucagon secretion from pancreatic α-cells. Glucagon is a hormone that
stimulates hepatic glucose production, especially during fasting or low blood glucose states.
Impact on Hepatic Glucose Production: By lowering glucagon levels, DPP-4 inhibitors reduce hepatic
glucose output, leading to lower blood glucose levels. This is particularly beneficial for postprandial
(after meal) glucose control.

4. Additional Effects:
Gastric Emptying: Increased GLP-1 levels can slow gastric emptying, contributing to improved
postprandial glucose levels and enhancing feelings of fullness (satiety).
Potential Cardiovascular Benefits: Some studies suggest that DPP-4 inhibitors may have beneficial
effects on cardiovascular health, although more research is needed to fully understand these effects.

Biguanides
Biguanides, primarily metformin, lower blood glucose levels through insulin-like effects on tissues. The
exact mechanism of action is not fully understood, but several key actions contribute to its effects:
Mechanism of Action (MOA)
1. Suppression of Hepatic Gluconeogenesis:
o AMP-Activated Protein Kinase (AMPK): Metformin primarily acts by activating AMPK,
which reduces glucose production in the liver.
 2. Inhibition of Intestinal Glucose Absorption:
o Metformin reduces the absorption of glucose from the intestines, contributing to lower
postprandial blood glucose levels.
3. Stimulation of Peripheral Glucose Uptake:
o In the presence of insulin, metformin enhances the uptake of glucose by peripheral
tissues, improving insulin sensitivity.
4. Stimulation of Glycolysis:
o Increases the breakdown of glucose within tissues, promoting energy production and
reducing blood glucose levels.
5. Reduction of Plasma Glucagon Levels:
o Lowering glucagon levels decreases hepatic glucose output, further aiding in blood
glucose control.
6. Decrease in Appetite:
o Metformin can lead to reduced appetite, which may assist in weight management.
Pharmacokinetics
 Absorption: Metformin is well-absorbed from the gastrointestinal tract.
 Duration of Action: 6–8 hours.
 Metabolism: Not metabolized; excreted unchanged in urine.
Adverse Effects
 Lactic Acidosis: While phenformin is associated with a high risk of lactic acidosis, metformin has
a much lower incidence. However, caution is necessary, especially in patients with renal or
hepatic dysfunction.
 Common Side Effects: Nausea, diarrhea, and a metallic taste; these are generally self-limiting.
 Vitamin B12 Deficiency: Long-term use may interfere with vitamin B12 absorption; B12 levels
should be monitored annually.
 Weight Management: Anorexia induced by metformin can be beneficial for weight loss.
Uses
 Diabetes Management: Primarily used in type 2 diabetes.
 Off-Label Uses: Metformin is also explored for conditions such as obesity, hirsutism, and
infertility in polycystic ovary syndrome (PCOS), where it can reduce androgen levels and
improve ovulation chances.
3. Thiazolidinediones (TZDs)
Overview: Thiazolidinediones (TZDs), also known as glitazones, are a class of antidiabetic medications
that act as agonists at the peroxisome proliferator-activated receptor gamma (PPAR-γ). These receptors
are nuclear receptors primarily located in adipose tissue, but also in muscle, liver, and other tissues.
Mechanism of Action (MOA)
 PPAR-γ Activation: TZDs activate PPAR-γ receptors, which modulate the expression of insulin-
sensitive genes. This activation leads to:
o Increased Insulin Sensitivity: Enhances insulin action in muscle and adipose tissues,
facilitating glucose transport.
o Glucose Utilization: Promotes the utilization of glucose, improving overall glucose
homeostasis.
o Reduction of Hepatic Gluconeogenesis: Lowers glucose production by the liver.
Advantages
 Dosing Convenience: Can be administered once daily.
 Low Hypoglycemia Risk: TZDs have a low potential for causing hypoglycemia when used alone.
 Lipid Profile Improvement: Increases HDL cholesterol levels.
 Minimal Drug Interactions: No significant drug interactions reported.
 Potential Mortality Reduction: Some studies suggest a reduction in all-cause mortality.
Disadvantages
  Time to Effect: It may take 6–12 weeks of treatment to reach maximum therapeutic effect,
requiring patience for optimal results.
 Weight Gain: Can cause weight gain due to increased fat storage.
 Cardiovascular Risks: Some concerns about heart failure and edema in certain populations.

4. α-Glucosidase Inhibitors
Overview: α-Glucosidase inhibitors, including acarbose, voglibose, and miglitol, are oral antidiabetic
agents that work by competitively inhibiting enzymes that digest carbohydrates in the intestines.
Mechanism of Action (MOA)

 Inhibition of α-Glucosidases: These enzymes are found in the intestinal brush border and are
responsible for breaking down disaccharides and oligosaccharides into monosaccharides (e.g.,
glucose and fructose) for absorption.
o Specific Enzymes: Acarbose inhibits enzymes such as sucrase, maltase, and
glycoamylase, while miglitol also inhibits isomaltase and beta-glucosidases.
 Effect on Carbohydrate Absorption:
o By inhibiting these enzymes, α-glucosidase inhibitors delay carbohydrate digestion and
reduce glucose absorption from the upper intestine.
o This leads to lower postprandial blood glucose levels, which is especially beneficial for
patients with postprandial hyperglycemia.
Advantages
 No Risk of Hypoglycemia: These medications do not cause hypoglycemia when used alone. If
used in combination with other antidiabetic agents and hypoglycemia occurs, glucose (not
sucrose) should be administered, as sucrose is also inhibited by the drug.
 Gastrointestinal Benefits: May be helpful for patients primarily experiencing postprandial
hyperglycemia.
Adverse Effects
 Gastrointestinal Disturbances: Common side effects include:
o Abdominal distention and pain
o Flatulence
o Diarrhea
 Mechanism of GI Effects: Undigested carbohydrates reach the colon, where they are fermented,
producing gas and fatty acids.
Contraindications
 Inflammatory Bowel Disease: Use is contraindicated due to potential exacerbation of symptoms.
 Renal Failure: Patients with renal impairment should avoid these medications.
Usage
 Monotherapy or Combination Therapy: Can be used alone or in combination with other oral
antidiabetic agents or insulin for improved glycemic control
Amylin Analogs
Overview: Amylin is a hormone produced by pancreatic beta cells that plays a crucial role in glucose
metabolism. It inhibits glucagon secretion, delays gastric emptying, and suppresses appetite. Pramlintide
is a synthetic analog of amylin that mimics these effects.
Mechanism of Action (MOA)

 Inhibition of Glucagon Secretion: Reduces postprandial glucose production by the liver.

 Delay of Gastric Emptying: Slows the rate at which food enters the small intestine, contributing
to a gradual rise in glucose levels.
 Appetite Suppression: Helps reduce food intake, aiding in weight management.
Pharmacokinetics
 Administration: Given subcutaneously.
 Absorption: Rapidly absorbed with a duration of action of approximately 2–3 hours.
 Dosing: Initiate with 15 µg before meals, with potential increase to 60–120 µg, adjusting
concurrent insulin doses to prevent hypoglycemia.
Adverse Effects
 Common side effects include nausea, vomiting, and anorexia.
 Should not be mixed with other medications in the syringe.
Indications
 Effective in both type 1 and type 2 diabetes mellitus.
SGLT-2 Inhibitors
Overview: Sodium-glucose cotransporter-2 (SGLT-2) inhibitors reduce glucose reabsorption in the
proximal tubule of the kidneys, leading to increased urinary glucose excretion (glycosuria).
Mechanism of Action (MOA)
 Inhibition of SGLT-2: Decreases reabsorption of glucose and sodium, resulting in glycosuria.
Common SGLT-2 Inhibitors
 Dapagliflozin
 Remogliflozin
 Canagliflozin
 Empagliflozin
 Sergliflozin
Benefits
 Particularly beneficial for patients with type 2 diabetes and hypertension.
 Low Hypoglycemia Risk: Generally do not cause hypoglycemia.
 Weight Loss: Associated with modest weight loss due to caloric loss from glucose.
Adverse Effects
 Potential for Hypotension: Due to diuretic effects.
 Increased risk of Urinary Tract Infections: Resulting from glucose in urine.
 Avoid in Low GFR: Contraindicated in patients with significantly reduced glomerular filtration
rate (GFR).
Glucagon
Overview: Glucagon is a peptide hormone produced by the alpha (A) cells of the pancreatic islets of
Langerhans. It plays a crucial role in glucose metabolism and energy regulation.
Synthesis and Regulation
 Synthesis: Produced in pancreatic alpha cells.
 Regulation: Secretion is influenced by:
o Nutrient levels, primarily glucose.
o Paracrine hormones.
 o Autonomic nervous system activity.
 Fasting Stimulus: Glucagon secretion is stimulated during fasting states.
Degradation
 Glucagon is metabolized in the liver, kidneys, and plasma.
Actions
 Increases Blood Glucose Levels:
o Glycogenolysis: Breaks down glycogen to glucose in the liver.
o Gluconeogenesis: Promotes the synthesis of glucose from non-carbohydrate sources.
 Insulin Release: Stimulates the release of insulin from pancreatic beta cells.
 Mobilization of Energy:
o Promotes the breakdown of stored fats and carbohydrates.
 Cardiac Effects: Increases heart rate and contractility.
 Smooth Muscle Relaxation: Relaxes intestinal smooth muscles.
Uses
1. Severe Hypoglycemia:
o Administered in emergency situations to treat severe hypoglycemia, especially due to
insulin overdose.
2. Diagnostic Tool:
o Used for diagnosing insulin-dependent diabetes mellitus (IDDM).
3. Radiology of the Bowel:
o Employed in imaging procedures, as it relaxes intestinal smooth muscles, aiding in better
visualization.
This concise summary highlights the key aspects of glucagon, including its synthesis, regulation, actions,
and clinical uses.

Oral Contraceptives
Definition: Oral contraceptives are medications taken by mouth to prevent pregnancy. They typically
contain hormones that regulate the menstrual cycle and inhibit ovulation.
Type Components Forms Administration
Combined Hormonal Estrogen + - Oral Pills: Monophasic, Daily (oral) / Monthly
Contraceptives (CHCs) Progestin Biphasic, Triphasic (injectable)
- Parenteral:
- Injectable Monthly
- Transdermal Patches Weekly
- Vaginal Rings Inserted for 3 weeks
Progestin-Only Progestin Only - Oral (Mini-Pill) Daily
Contraceptives (POCs) - Parenteral:
- IM Injections Every 3 months
- Subcutaneous Implants Up to 5 years
Postcoital Contraceptives Estrogen + - Emergency Contraceptive Within 72-120 hours
Progestin / Pills after intercourse
Progestin Only - Intrauterine Devices Inserted within 5 days
(IUDs)

1. Combined Hormonal Contraceptives (CHCs)

Mechanism of Action (MOA):
 Inhibition of Ovulation: Estrogen suppresses the release of Follicle-Stimulating Hormone (FSH)
from the pituitary gland, preventing the development of ovarian follicles. Progestin inhibits
Luteinizing Hormone (LH) release, blocking the LH surge necessary for ovulation.
  Cervical Mucus Alteration: Progestin increases the viscosity of cervical mucus, creating a barrier
to sperm penetration.
 Endometrial Changes: Progestin induces changes in the endometrial lining, making it less
receptive to implantation, should fertilization occur.
 Altered Tubal Motility: OCs may affect the contractions of the fallopian tubes, reducing sperm
transport and ovum movement.
Pharmacology:
 Absorption and Metabolism: Typically administered orally; well-absorbed in the gastrointestinal
tract. The liver metabolizes these hormones, and they undergo first-pass metabolism.
 Duration of Action: Effective throughout the menstrual cycle when taken as directed.
 Impact on Lipid Metabolism: Can lead to increased levels of high-density lipoprotein (HDL)
cholesterol and changes in triglycerides.
Adverse Drug Reactions (ADRs):
 Common (Mild): Nausea, vomiting, headaches, breast tenderness, mood changes, and weight
gain.
 Serious (Severe):
o Thromboembolic Events: Increased risk of venous thrombosis and pulmonary
embolism, particularly in women over 35, smokers, or those with other risk factors.
o Cardiovascular Issues: Potential for hypertension and other cardiovascular
complications.
o Gallbladder Disease: Increased risk in some women.
Uses:
 Primary Use: Prevention of pregnancy.
 Menstrual Regulation: Treatment for menstrual irregularities, dysmenorrhea, and menorrhagia.
 Non-contraceptive Benefits: Can reduce the risk of ovarian and endometrial cancers, improve
acne, and manage polycystic ovary syndrome (PCOS).

2. Progestin-Only Contraceptives (POCs)

Mechanism of Action (MOA):
 Inhibition of Ovulation: Although less reliable than CHCs, POCs can prevent ovulation in some
women.
 Cervical Mucus Thicken: Progestin increases mucus viscosity, which obstructs sperm entry into
the uterus.
 Endometrial Modifications: Progestin alters the endometrial lining to make it less favorable for
implantation.
Pharmacology:
 Absorption: Administered orally or via injections and implants; provides continuous release of
progestin.
 Effects on Menstrual Cycle: May lead to irregular bleeding patterns; some users may experience
amenorrhea (absence of menstruation) over time.
Adverse Drug Reactions (ADRs):
 Common (Mild): Irregular menstrual cycles, headaches, breast tenderness, mood swings.
 Long-Term Concerns: Prolonged use may lead to decreased bone density, particularly with
injectable forms.
Uses:
 Alternative for Estrogen-Sensitive Patients: Suitable for women who cannot tolerate estrogen
(e.g., breastfeeding mothers).
 Long-Term Contraception: Effective for extended periods, particularly with implantable and
injectable forms.

3. Postcoital Contraceptives
Mechanism of Action (MOA):
 Prevent Ovulation: If taken before ovulation, they inhibit or delay ovulation through
suppression of the hypothalamic-pituitary-ovarian axis.
 Alter Endometrial Lining: Changes in the uterine lining can prevent implantation if ovulation
has already occurred.
Pharmacology:
 Administration: Oral pills are taken in high doses within 72 to 120 hours post-intercourse;
effectiveness decreases over time.
 First-Pass Metabolism: Similar to CHCs, these drugs undergo first-pass metabolism, influencing
their bioavailability.
Adverse Drug Reactions (ADRs):
 Common (Mild): Nausea, vomiting, fatigue, dizziness, headache, and breast tenderness.
 Menstrual Changes: Can cause irregular bleeding or changes in the menstrual cycle following
use.
Uses:
 Emergency Contraception: Intended for use after unprotected intercourse or contraceptive
failure.

Bioassay of Insulin
Insulin is a peptide hormone produced by the pancreatic β cells, crucial for glucose metabolism. It is
initially synthesized as proinsulin and then processed to its active form within secretory granules.
Bioassays are essential for assessing the biological activity and potency of insulin preparations.
Methods of Bioassay
1. Rabbit Method
Selection of Rabbits:
 Criteria: Use healthy rabbits weighing 1800-3000 grams.
 Preparation: Maintain on a uniform diet and fast for 18 hours before the assay. Water should also
be withheld during the experiment.
Standard Preparation:
 Preparation of Standard Solution:
o Weigh 20 units of pure insulin.
o Dissolve in normal saline.
o Acidify to pH 2.5 using hydrochloric acid.
o Add 0.5% phenol as a preservative and 1.4% to 1.8% glycerin to stabilize the solution.
o The final concentration should be 20 units/ml.
o Store in a cool place and use within six months.
Experimental Procedure:
1. Initial Blood Sugar Measurement:
o Take a blood sample from the marginal ear vein of each rabbit to determine the baseline
blood glucose level.
2. Standard and Sample Dilutions:
o Prepare standard dilutions containing 1 unit/ml and 2 units/ml in normal saline.
3. Dosing Groups:
o Divide rabbits into four groups (3 rabbits each):
Group Dilution Number of Purpose
Animals
Group I Standard dilution (1 3 rabbits Assess hypoglycemic effect of standard insulin.
unit/ml)
Group Standard dilution (2 3 rabbits Assess hypoglycemic effect of higher
II units/ml) concentration of standard insulin.
 Group Test dilution (1 3 rabbits Evaluate hypoglycemic effect of test insulin
III unit/ml) sample.
Group Test dilution (2 3 rabbits Evaluate hypoglycemic effect of higher
IV units/ml) concentration of test insulin sample.
4. Injection and Observation:
o Initial Injection: Administer subcutaneous injections of the assigned insulin dilutions.
o Final Blood Sugar Measurement: Blood samples are taken at 1-hour intervals for up to 5
hours post-injection to determine the "Final Blood Sugar Level."
5. Cross-Over Test:
o After one week, repeat the test using the same rabbits but reverse the groups to eliminate
variability. Measure blood glucose levels again, and calculate the mean percentage
decrease in blood sugar from both tests.

2. Mouse Method
Selection of Mice:
 Criteria: Use a minimum of 100 mice, each weighing 18-22 grams. Maintain on a constant diet
and fast for 18 hours prior to the experiment.
Standard Preparation:
 Preparation of Standard Solution:
o Prepare dilutions containing 0.064 units/ml (standard dilution I) and 0.096 units/ml
(standard dilution II) in sterile saline.
Experimental Procedure:
1. Grouping: Divide mice into four groups (25 mice each):
Group Dilution Number of Purpose
Animals
Group I Standard dilution 25 mice Compare convulsion rate induced by low
(0.064 units/ml) concentration of standard insulin.
Group Standard dilution 25 mice Compare convulsion rate induced by higher
II (0.096 units/ml) concentration of standard insulin.
Group Test dilution 25 mice Assess convulsion rate induced by test insulin
III sample.
Group Test dilution 25 mice Assess convulsion rate induced by higher
IV concentration of test insulin sample.
2. Injection and Observation:
o Injection: Administer subcutaneous injections of the assigned insulin dilutions.
o Incubation: Place mice in an incubator set to 33°C for 1.5 hours.
o Observation for Convulsions: Monitor for convulsions or failure to right themselves,
which indicates insulin activity. Convulsive mice can be rescued with a 0.5 ml injection of
5% dextrose solution.
3. Analysis:
o Calculate the percentage of convulsions produced by the test samples compared to the
standard samples. Mice that survive can be reused for further tests after an interval of
one week.
Calculating Insulin Activity
Potency Assessment:
 Mean Percentage Decrease: For the rabbit method, calculate the mean percentage decrease in
blood glucose between the initial and final measurements for each group.
 Convulsion Percentage: In the mouse method, compare the percentage of convulsions induced
by the test samples against the standards.
Considerations
  Standardization: Ensure that all insulin preparations are standardized to maintain consistency
across tests.
 Ethical Guidelines: Follow ethical protocols for animal research to minimize suffering and
adhere to humane treatment standards.
Applications
 Quality Control: This bioassay is critical for quality control of insulin products in pharmaceutical
manufacturing.
 Research Applications: The methods are also valuable in research settings for studying the
physiological effects of insulin, diabetes pathophysiology, and drug interactions.
This detailed bioassay procedure provides a reliable method for evaluating insulin's biological activity,
essential for ensuring the efficacy and safety of insulin formulations used in clinical practice.

Bioassay of Oxytocin
Overview
Oxytocin is a peptide hormone and neuropeptide produced by the paraventricular nucleus of the
hypothalamus and released by the posterior pituitary. It plays a crucial role in reproductive processes,
including inducing uterine contractions during childbirth and controlling postpartum bleeding. Oxytocin
is also utilized to stimulate uterine contractions in cases of incomplete or threatened miscarriage.
Bioassay Methods
Different bioassay methods for assessing oxytocin activity include:
 Contraction of the rat uterus
 Depression of blood pressure in chickens
 Measurement of milk-ejection pressure in lactating rats
Principle
The potency of an oxytocin injection is determined by comparing its activity to that of a standard
preparation of oxytocin under specified assay conditions.
Standard Preparation
 Composition: A freeze-dried preparation of oxytocin combined with human albumin and citric
acid (available in ampoules containing 12.5 units).
 Unit Definition: The specific oxytocin activity corresponding to 0.0005 g of the standard
preparation.
 International Standard: Based on the 4th international standard for oxytocin established in 1978.

Experimental Methods
1. By Contraction of the Rat Uterus
Selection Criteria:
 Use female rats weighing between 120-200 grams.
 Confirm that the rat is in estrous or pre-estrous through vaginal smear.
Procedure:
1. Pre-treatment: Inject 100 µg of estradiol benzoate intramuscularly 18-24 hours before the assay.
2. Uterus Preparation: Kill the rat and suspend one horn of the uterus in a bath containing:
o Sodium chloride: 0.662%
o Potassium chloride: 0.045%
o Calcium chloride: 0.007%
o Sodium bicarbonate: 0.256%
o Disodium hydrogen phosphate: 0.029%
o Sodium dihydrogen phosphate: 0.003%
o Magnesium chloride: 0.010%
o Dextrose: 0.050%
3. Bath Maintenance:
o Maintain at 32°C to abolish spontaneous contractions.
o Oxygenate with a mixture of 95% oxygen and 5% carbon dioxide.
 4. Dosing and Recording:
o Add two doses of the standard preparation (10-50 micro units/ml) to the bath, ensuring
clearly discriminated, submaximal contractions.
o Replace the bath liquid after maximal contraction is reached.
o Maintain a constant ratio of doses between the test and standard preparations.
o Record at least six responses using a randomized order or Latin square design, and
analyze results statistically.

2. By Depression of Blood Pressure in Chickens

Selection Criteria:
 Use healthy young adult cockerels weighing 1.2 to 2.3 kg.
Procedure:
1. Anesthesia: Anesthetize using an agent that maintains a constant high blood pressure.
2. Surgical Preparation: Expose the gluteus muscle, cannulate the popliteal artery, and record
blood pressure.
3. Injection Setup:
o Prepare standard solution with saline (0.1-0.5 ml for injection).
o Administer two doses of the standard solution (20-100 milli units) via the cannulated
vein, allowing for a constant interval (3-10 minutes) between injections.
4. Dosing and Recording:
o Dilute the test sample with saline to match standard preparation responses.
o Maintain the same dosing ratio throughout the assay.
o Use a randomized order for administration and record at least six responses, analyzing
results statistically.

3. By Measurement of Milk-Ejection Pressure in Lactating Rats

Selection Criteria:
 Use lactating rats (3-21 days postpartum) weighing about 300 g.
Procedure:
1. Anesthesia: Anesthetize the rat using pentobarbitone sodium.
2. Cannulation:
o Cannulate the trachea and a jugular or femoral vein with polyethylene tubes.
o Cannulate one inguinal teat to measure pressure.
3. Setup:
o Connect the teat cannula to a strain gauge transducer, filling the system with a sodium
citrate or saline solution containing heparin to prevent clotting.
4. Dosing and Recording:
o Inject solutions through the venous cannula (0.1-0.4 ml).
o Choose doses of the standard preparation to achieve specified increases in milk-ejection
pressure (about 1.35 kPa for the lower dose and about 2.7 kPa for the higher dose).
o Maintain a consistent inter-dose ratio for the test preparation and administer at intervals
of 3-5 minutes, recording at least four responses for statistical analysis.

Considerations
 Standardization: Ensure that all oxytocin preparations are standardized for consistency across
tests.
 Ethical Guidelines: Follow ethical protocols for animal research to minimize suffering and
ensure humane treatment.
Applications
 Quality Control: Essential for quality control of oxytocin products in pharmaceutical
manufacturing.
  Research Applications: Valuable for studying physiological effects of oxytocin and its role in
reproductive health.

Bioassay of Histamine
Overview
Histamine is a biogenic amine involved in various physiological functions, including immune response,
gastric acid secretion, and neurotransmission. Its biological effects are mediated through specific
histamine receptors (H1, H2, H3, H4), and its measurement is important for both research and clinical
applications.
Bioassay Methods
Common bioassay methods for assessing histamine activity include:
 Contraction of the Isolated Uterus or Intestinal Muscle
 Vasodilation in the Perfused Rabbit Ear
 Measurement of Gastric Secretion in Rats
Principle
The potency of histamine preparations is determined by comparing their effects on specific biological
tissues to a standard histamine preparation under controlled conditions.
Standard Preparation
 Composition: A standard histamine solution prepared in a suitable solvent (e.g., saline).
 Unit Definition: Histamine activity is typically expressed in terms of the response elicited in a
biological system (e.g., contraction or vasodilation).
 International Standard: Based on established guidelines for histamine activity.
Experimental Methods
1. Contraction of the Isolated Uterus or Intestinal Muscle
Selection Criteria
 Use female rats or guinea pigs for uterine assays, or appropriate models for intestinal assays.
Procedure
1. Preparation: Euthanize the animal and isolate the uterus or a segment of the intestine.
2. Bath Setup: Suspend the tissue in a physiological saline bath, maintaining a temperature of 37°C
and oxygenation.
3. Dosing and Recording:
o Administer varying doses of the histamine standard (e.g., 0.1–1.0 µg/ml).
o Record the contractions or relaxation responses.
o Use a randomized order for administration and conduct multiple trials for statistical
analysis.
2. Vasodilation in the Perfused Rabbit Ear
Selection Criteria
 Use healthy young adult rabbits.
Procedure
1. Anesthesia: Anesthetize the rabbit with an appropriate agent.
2. Surgical Preparation: Cannulate the artery supplying the ear and connect it to a perfusion
system.
3. Dosing and Recording:
o Administer a series of histamine doses (e.g., 0.1–1.0 mg) via the cannulated artery.
o Measure changes in ear blood flow or diameter as an indication of vasodilation.
o Use a consistent dosing interval and record multiple responses for analysis.
3. Measurement of Gastric Secretion in Rats
Selection Criteria
 Use healthy adult rats.
Procedure
1. Anesthesia: Anesthetize the rat using an appropriate method (e.g., ketamine).
2. Cannulation: Cannulate the stomach to collect gastric secretions.
 3. Dosing and Recording:
o Inject standard histamine doses (e.g., 0.5–5.0 mg/kg) via the intraperitoneal route.
o Collect gastric secretions for a fixed period post-injection.
o Measure volume and acidity as indicators of histamine activity.
Considerations
 Standardization: Ensure all histamine preparations are standardized for consistency across
assays.
 Ethical Guidelines: Follow ethical protocols for animal research to ensure humane treatment.
Applications
 Pharmaceutical Testing: Essential for evaluating the efficacy of antihistamines and other related
drugs.
 Research Applications: Valuable for studying the role of histamine in various physiological and
pathological processes.
Bioassay of Digitalis
Overview
Digitalis refers to a group of medications derived from the leaves of the Digitalis purpurea (foxglove)
plant, primarily used in the treatment of heart conditions, particularly heart failure and atrial fibrillation.
The active compounds, known as cardiac glycosides (e.g., digoxin), increase the force of cardiac
contractions and regulate heart rhythm.
Bioassay Methods
Several bioassay methods can be employed to assess the activity of digitalis compounds, including:
 Contraction of Isolated Heart Muscle (e.g., Frog Heart Assay)
 In Vivo Studies in Small Animals
 Measurement of Cardiac Output in Isolated Perfused Hearts
Principle
The potency of digitalis preparations is assessed by comparing their effects on cardiac muscle contraction
or other cardiovascular responses to a standard digitalis preparation under controlled conditions.
Standard Preparation
 Composition: A standardized extract of digitalis leaves, typically containing digoxin or other
cardiac glycosides.
 Unit Definition: Activity is expressed in terms of a specific concentration (e.g., nanograms per
milliliter) that produces a defined physiological effect.
 International Standard: Based on established standards for digitalis potency.
Experimental Methods
1. Contraction of Isolated Heart Muscle
Selection Criteria
 Use adult frogs or similar small animals for isolated heart assays.
Procedure
1. Preparation: Euthanize the animal and remove the heart.
2. Bath Setup: Place the heart in a physiological saline solution at 25-30°C, ensuring it is
oxygenated.
3. Dosing and Recording:
o Administer varying doses of the standard digitalis preparation (e.g., 1-10 µg/ml).
o Record the force and rate of heart contractions using a force transducer.
o Analyze multiple responses for statistical significance.
2. In Vivo Studies in Small Animals
Selection Criteria
 Use healthy adult rodents, such as rats or mice.
Procedure
1. Anesthesia: Anesthetize the animal using an appropriate agent.
2. Dosing: Administer a series of digitalis doses (e.g., 0.1-1.0 mg/kg) via the intravenous or
intraperitoneal route.
 3. Monitoring:
o Continuously monitor heart rate and rhythm using an ECG.
o Record any changes in cardiac output or contractility.
3. Measurement of Cardiac Output in Isolated Perfused Hearts
Selection Criteria
 Use isolated hearts from small mammals, such as rats or guinea pigs.
Procedure
1. Preparation: Euthanize the animal and isolate the heart.
2. Perfusion Setup: Mount the heart in a perfusion apparatus with a suitable physiological solution.
3. Dosing and Recording:
o Administer digitalis preparations at varying concentrations.
o Measure cardiac output and contractility using appropriate instruments.
o Record multiple responses for analysis.
Considerations
 Standardization: Ensure all digitalis preparations are standardized for accurate comparisons.
 Ethical Guidelines: Follow ethical protocols to minimize animal suffering and ensure humane
treatment.
Applications
 Pharmaceutical Development: Critical for testing the efficacy and safety of new cardiac
glycoside formulations.
 Clinical Research: Valuable for studying the pharmacodynamics and pharmacokinetics of
digitalis compounds in heart disease management.

Drugs that act on the uterus play a crucial role in managing various reproductive health issues and are
essential in obstetrics and gynecology. These medications can influence uterine contractions, hormone
levels, and overall uterine function, providing therapeutic benefits in a range of clinical scenarios.
The classification of these drugs is based on their pharmacological effects and intended uses. Uterotonics,
for instance, are utilized to induce labor or control postpartum hemorrhage, while tocolytics are
employed to inhibit premature contractions.
Classification of drugs acting on the uterus:
Category Drug Examples Mechanism of Action Therapeutic Uses
Uterotonics - Oxytocin Stimulates uterine Induction of labor,
contractions control postpartum
hemorrhage
- Prostaglandins (e.g., Induces uterine Labor induction,
Misoprostol, Dinoprostone) contractions and medical abortion
cervical ripening
- Ergot Alkaloids (e.g., Causes sustained Prevents postpartum
Ergometrine) uterine contractions hemorrhage
Tocolytics - Beta-Agonists (e.g., Relaxes uterine smooth Delays premature
Terbutaline) muscle labor
- Calcium Channel Blockers Inhibits calcium influx, Delays premature
(e.g., Nifedipine) leading to muscle labor
relaxation
- Magnesium Sulfate Acts as a smooth Delays premature
muscle relaxant labor
Hormonal Agents - Progestins (e.g., Maintains pregnancy Prevents premature
Medroxyprogesterone by modulating contractions
acetate) hormonal activity
 - Estrogens Regulates menstrual Hormone replacement
cycle and uterine therapy
growth
Antiprogestins - Mifepristone (RU-486) Blocks progesterone Induces abortion
receptors
Antiinflammatories - NSAIDs (e.g., Ibuprofen, Reduces pain and Manages
Naproxen) inflammation dysmenorrhea
Other Agents - Danazol Suppresses estrogen Treats endometriosis
and progesterone
production
- Leuprolide Reduces estrogen Manages
levels by acting as a endometriosis and
GnRH analog uterine fibroids

2 Marks
1. Drugs Used in Thyroid Disorders
 Levothyroxine: Synthetic T4 used to treat hypothyroidism.
 Liothyronine: Synthetic T3 for severe hypothyroidism.
 Methimazole: Antithyroid drug used to treat hyperthyroidism by inhibiting thyroid hormone
synthesis.
 Propylthiouracil (PTU): Antithyroid medication that blocks hormone production and conversion
of T4 to T3.
 Radioactive Iodine: Used for treating hyperthyroidism by destroying overactive thyroid cells.
2. Synthetic Progestins
 Medroxyprogesterone Acetate: Used in hormone replacement therapy and contraception.
 Norethindrone: Commonly used in contraceptives and for menstrual disorders.
 Desogestrel: A progestin in some oral contraceptives that helps regulate menstrual cycles.
 Levonorgestrel: Used in emergency contraception and hormonal IUDs.
3. Oxytocics
 Oxytocin: Induces labor and controls postpartum bleeding by stimulating uterine contractions.
 Methylergometrine: Used to prevent postpartum hemorrhage by promoting uterine contraction.
 Carbetocin: Similar to oxytocin, used to reduce bleeding after cesarean sections.
4. Glucocorticoids
 Hydrocortisone: Used to treat adrenal insufficiency and inflammatory conditions.
 Prednisone: Commonly prescribed for autoimmune diseases and allergies.
 Dexamethasone: Used for its anti-inflammatory effects in various conditions, including severe
allergies and asthma.
5. Anabolic Steroids
 Testosterone: Used for hormone replacement therapy and to promote muscle growth.
 Nandrolone: Used in certain medical conditions and for muscle-building in athletes.
 Stanozolol: Used for its anabolic properties, primarily in veterinary medicine and bodybuilding.