EXPERIMENT#1 Title: To Demonstrate the Different Stages/Phases of Drug Discovery and Development: Any drug development process must proceed through several stages in order to produce a product that is safe, efficacious, and has passed all regulatory requirements. Detailed Stages of Drug Discovery and Development: Discovery Phase: 1. Pre-discovery 2. Discovery of the Lead. 3. Early Safety Tests. 4. Lead Optimization. 5. Preclinical Testing. Development Phase: 1. IND Application and Safety. 2. Clinical Trials. 3. NDA and Approval. 4. Manufacturing. 5. Post marketing Surveillance and Phase 4 trials 1. Pre-discovery: This stage consists of the following steps: a. Understand the disease: Before any potential new medicine can be discovered, scientists work to understand the disease to be treated as well as to unravel the underlying cause of the condition. They try to understand how the genes are altered, how that affects the proteins they encode and how those proteins interact with each other in living cells, how those affected cells change the specific tissue they are in and finally how the disease affects the entire patient. This knowledge is the basis for treating the problem. Researchers from government, academia and industry all contribute to this knowledge base. However, even with new tools and insights, this research takes many years of work and, too often, leads to frustrating dead ends. And even if the research is successful, it will take many more years of work to turn this basic understanding of what causes a disease into a new treatment.
b. Target Identification (Choose a molecule to target with a drug): Once they have enough understanding of the underlying cause of a disease, pharmaceutical researchers select a target for a potential new medicine. A target is generally a single molecule, such as a gene or protein, which is involved in a particular disease. Even at this early stage in drug discovery it is critical that researchers pick a target that is drugable, i.e., one that can potentially interact with and be affected by a drug molecule. c. Target Validation (Test the target and confirm its role in the disease) After choosing a potential target, scientists must show that it actually is involved in the disease and can be acted upon by a drug. Target validation is crucial to help scientists avoid research paths that look promising, but ultimately lead to dead ends. Researchers demonstrate that a particular target is relevant to the disease being studied through complicated experiments in both living cells and in animal models of disease. Discovery (Find a promising molecule (a lead compound) that could become a drug): Armed with their understanding of the disease, scientists are ready to begin looking for a drug. They search for a molecule, or lead compound, that may act on their target to alter the disease course. Up to 5,000 to 10,000 molecules for each potential drug candidate are subjected to a rigorous screening process. Once scientists confirm interaction with the drug target, they typically validate that target by checking for activity versus the disease condition for which the drug is being developed. After careful review, one or more lead compounds are chosen. If successful over long odds and years of testing, the lead compound can ultimately become a new medicine. 2. 3. Early Safety Tests (Perform initial tests on promising compounds):
Lead compounds go through a series of tests to provide an early assessment of the safety of the lead compound. Scientists test Absorption, Distribution, Metabolism, Excretion and Toxicological (ADME/Tox) properties, or pharmacokinetics, of each lead. Successful drugs must be: absorbed into the bloodstream, distributed to the proper site of action in the body, metabolized efficiently and effectively, successfully excreted from the body and demonstrated to be not toxic. These studies help researchers prioritize lead compounds early in the discovery process. ADME/Tox studies are performed in living cells, in animals and via computational models.
Lead Optimization (Alter the structure of lead candidates to improve properties):
Lead compounds that survive the initial screening are then optimized, or altered to make them more effective and safer. By changing the structure of a compound, scientists can give it different properties. For example, they can make it less likely to interact with other chemical pathways in the body, thus reducing the potential for side effects. Hundreds of different variations or analogues of the initial leads are made and tested. Teams of biologists and chemists work together closely: The biologists test the effects of analogues on biological systems while the chemists take this information to make additional alterations that are then retested by the biologists. The resulting compound is the candidate drug. Even at this early stage, researchers begin to think about how the drug will be made, considering formulation (the recipe for making a drug, including inactive ingredients used to hold it together and allow it to dissolve at the right time), delivery mechanism (the way the drug is taken by mouth, injection, inhaler) and largescale manufacturing (how you make the drug in large quantities).
Preclinical Testing (Lab and animal testing to determine if the drug is safe enough for human testing): With one or more optimized compounds in hand, researchers turn their attention to testing them extensively to determine if they should move on to testing in humans. Scientists carry out in vitro and in vivo tests. In vitro tests are experiments conducted in the lab, usually carried out in test tubes and beakers (vitro is glass in Latin) and in vivo studies are those in living cell cultures and animal models (vivo is life in Latin). Scientists try to understand how the drug works and what its safety profile looks like. The U.S. Food and Drug Administration (FDA) requires extremely thorough testing before the candidate drug can be studied in humans. During this stage researchers also must work out how to make large enough quantities of the drug for clinical trials. Techniques for making a drug in the lab on a small scale do not translate easily to larger production. This is the first scale up. The drug will need to be scaled up even more if it is approved for use in the general patient population. At the end of several years of intensive work, the discovery phase concludes. After starting with approximately 5,000 to 10,000 compounds, scientists now have winnowed the group down to between one and five molecules, candidate drugs, which will be studied in clinical trials. The Development Process/Phase: 1. Investigational New Drug (IND) Application and Safety: (File IND with the FDA before clinical testing can begin; ensure safety for clinical trial volunteers through an Institutional Review Board); Before any clinical trial can begin, the researchers must file an Investigational New Drug (IND) application with the FDA. The application includes the results of the preclinical work, the3
candidate drugs chemical structure and how it is thought to work in the body, a listing of any side effects and manufacturing information. The IND also provides a detailed clinical trial plan that outlines how, where and by whom the studies will be performed. The FDA reviews the application to make sure people participating in the clinical trials will not be exposed to unreasonable risks. In addition to the IND application, all clinical trials must be reviewed and approved by the Institutional Review Board (IRB) at the institutions where the trials will take place. This process includes the development of appropriate informed consent, which will be required of all clinical trial participants. Statisticians and others are constantly monitoring the data as it becomes available. The FDA or the sponsor company can stop the trial at any time if problems arise. In some cases a study may be stopped because the candidate drug is performing so well that it would be unethical to withhold it from the patients receiving a placebo or another drug. Finally, the company sponsoring the research must provide comprehensive regular reports to the FDA and the IRB on the progress of clinical trials. 2. Clinical Trials:
Phase 1 Clinical Trial (Perform initial human testing in a small group of healthy volunteers): In Phase 1 trials the candidate drug is tested in people for the first time. These studies are usually conducted with about 20 to 100 healthy volunteers. The main goal of a Phase 1 trial is to discover if the drug is safe in humans. Researchers look at the pharmacokinetics of a drug: How is it absorbed? How is it metabolized and eliminated from the body? They also study the drugs pharmacodynamics: Does it cause side effects? Does it produce desired effects? These closely monitored trials are designed to help researchers determine what the safe dosing range is and if it should move on to further development. Phase 2 Clinical Trial (Test in a small group of patients): In Phase 2 trials researchers evaluate the candidate drugs effectiveness in about 100 to 500 patients with the disease or condition under study, and examine the possible short-term side effects (adverse events) and risks associated with the drug. They also strive to answer these questions: Is the drug working by the expected mechanism? Does it improve the condition in question? Researchers also analyze optimal dose strength and schedules for using the drug. If the drug continues to show promise, they prepare for the much larger Phase 3 trials. Phase 3 Clinical Trial (Test in a large group of patients to show safety and efficacy): In Phase 3 trials researchers study the drug candidate in a larger number (about 1,000-5,000) of patients to generate statistically significant data about safety, efficacy and the overall benefit-risk4
relationship of the drug. This phase of research is key in determining whether the drug is safe and effective. It also provides the basis for labeling instructions to help ensure proper use of the drug (e.g., information on potential interactions with other medicines). Phase 3 trials are both the costliest and longest trials. Hundreds of sites around the United States and the world participate in the study to get a large and diverse group of patients. Coordinating all the sites and the data coming from them is a monumental task. During the Phase 3 trial (and even in Phases 1 and 2), researchers are also conducting many other critical studies, including plans for full scale production and preparation of the complex application required for FDA approval.
New Drug Application (NDA) and Approval (Submit application for approval to FDA): Once all three phases of the clinical trials are complete, the sponsoring company analyzes all of the data. If the findings demonstrate that the experimental medicine is both safe and effective, the company files a New Drug Application (NDA) which can run 100,000 pages or more with the FDA requesting approval to market the drug. The NDA includes all of the information from the previous years of work, as well as the proposals for manufacturing and labeling of the new medicine. FDA experts review all the information included in the NDA to determine if it demonstrates that the medicine is safe and effective enough to be approved (see sidebar How does the FDA decide to approve a new drug?). Following rigorous review, the FDA can either 1) approve the medicine, 2) send the company an approvable letter requesting more information or studies before approval can be given, or 3) deny approval. Review of an NDA may include an evaluation by an advisory committee, an independent panel of FDA-appointed experts who consider data presented by company representatives and FDA reviewers. Committees then vote on whether the FDA should approve an application, and under what conditions. The FDA is not required to follow the recommendations of the advisory committees, but often does. 4. Manufacturing: Going from small-scale to large-scale manufacturing is a major undertaking. In many cases, companies must build a new manufacturing facility or reconstruct an old one because the manufacturing process is different from drug to drug. Each facility must meet strict FDA guidelines for Good Manufacturing Practices (GMP). Making a high-quality drug compound on a large scale takes great care. Imagine trying to make a cake, for example, on a large scale making sure the ingredients are evenly distributed in the mix, ensuring that it heats evenly. The process to manufacture most drugs is even more complicated than this. There are few, if any, other businesses that require this level of skill in manufacturing.
5. Ongoing/ Post Marketing Studies and Phase 4 Trials: Research on a new medicine continues even after approval. As a much larger number of patients begin to use the drug, companies must continue to monitor it carefully and submit periodic reports, including cases of adverse events, to the FDA. In addition, the FDA sometimes requires a company to conduct additional studies on an approved drug in Phase 4 studies. These trials can be set up to evaluate long-term safety or how the new medicine affects a specific subgroup of patients.
EXPERIMENT#2 Title: To Demonstrate Different Requirements/Considerations for Dosage Form Design: Following are the factors which are considered in the design of a pharmaceutical dosage form: 1. 2. 3. 4. 5. Biopharmaceutical Considerations. Physicochemical Considerations. Processing Considerations. Marketing Considerations. Regulatory Considerations.
1. Biopharmaceutical Considerations: Mechanism of Action Target Organ Dose (Potency) Permeability (Passive, Active, Efflux) Pharmacokinetics: Absorption Distribution Metabolism Excretion.
Physical/Chemical Considerations: Solubility (Dissolution) log P, PSA, H-donors, H-acceptors mw Stability (Heat, Humidity, Light) pH Excipient Compatibility Morphology Density (Bulk and Tap) Particle Size Wetting (Surface Energy) Static Properties Flow Properties Compressibility and Compactability Hygroscopicity Polymorphism
Processing Considerations: Cost of Goods Capital Investments Dosage Form (Tablet vs. Hard Gelatin Capsules) Size (6mm Tablet vs. 11mm Tablet) Shape (Round Tablet vs. Unique Shaped -Keyed Tools) Excipients (Alternate Suppliers) Processing Efficiency (Number of Process Steps, Speed of Processing, Volume of Process) Failure Rate (Rejected Batches) Marketing Considerations: Time for Development Patent Protection Competitive Advantage Aesthetics (Size, Shape, Color, Taste, Painless) Patient Compliance (Once-A-Day vs. bid) Price (Cost of Goods)
Regulatory Considerations: Documented Formula, Process and Packaging Active Ingredients Excipients Testing Methods Specifications Equipment Unit Operations In-Process Controls Validation of the Process Validation of the Release and Stability Testing Methods Stability Report on Package Drug Product (Bulk, Primary) Documents IND, NDAs, Validation Reports
EXPERIMENT#3 Title: To Determine the Bulk Densities of the Given Powders: Bulk Density: Apparent (or bulk or Scott density) is the density of a weight of a certain volume of powder. The powder is allowed to flow freely and gently into a container of a known volume. (or) Bulk density is the mass of a given volume of a powder The total volume includes particle volume, inter-particle void volume and internal pore volume. It is determined by dividing the total mass of a given volume of the powder. Requirements: NaCl, Starch, Kaolin, Talc, Graduated cylinder, weighing balance. Procedure: Weigh the graduated cylinder (100ml), and then pour the powder by means of a funnel into a 100 ml graduated cylinder up to the mark. Weigh the filled cylinder and then subtract the weight of empty cylinder from that of filled cylinder to determine the mass of the powder (m). Finally determine the bulk density of the powder by dividing the mass of the powder by its volume. Bulk density = m/v m = mass of the powder v = volume of the powder (100 ml) Bulk density is measured in the units of g/ml.
EXPERIMENT#4 Title: To Determine the Tape Densities of the Given Powders: Tape Density: Tap density (or packed density) refers to the volume of a specific weight of powder after it has been settled or packed until no further volume change is observed. Tapped density is achieved by mechanically tapping a measuring cylinder containing a powder sample. After observing the initial volume, the cylinder is mechanically tapped, and volume readings are taken until little further volume change is observed. The mechanical tapping is achieved by raising the cylinder and allowing it to drop under its own weight a specified distance. The difference is that the tap density is the density of a compacted powder and bulk density is the density of a powder with no compaction at all. Requirements: NaCl, Starch, Kaolin, Talc, Graduated cylinder, weighing balance. Procedure: Weigh the graduated cylinder (100ml), and then pour the powder by means of a funnel into a 100 ml graduated cylinder up to the mark and record this volume as Vo. Weigh the filled cylinder and then subtract the weight of empty cylinder from that of filled cylinder to determine the mass of the powder. Mechanically tap the cylinder containing the sample by raising the cylinder and allowing it to drop under its own weight. Continue the tapping for 1000 times or until there is no further reduction in the volume. Determine the volume after tapping as Vf. Finally determine the tapped density of the powder by dividing the mass of the powder by its final volume. Tapped density = m/Vf m = mass of the powder Vf = volume of the powder after tapping Tapped density is measured in the units of g/ml.
EXPERIMENT#5 Title: To Determine the Compressibility Index of the Given Powders and to Characterize These Powders on the Basis of the Compressibility Index: The Compressibility Index is measures of the propensity of a powder to be compressed. As such, it is the measures of the relative importance of inter particulate interactions. In a free-flowing powder, such interactions are generally less significant, and the bulk and tapped densities will be closer in value. For poorer flowing materials, there are frequently greater inter particle interactions, and a greater difference between the bulk and tapped densities will be observed. These differences are reflected in the Compressibility Index. Requirements: NaCl, Starch, Kaolin, Talc, Graduated cylinder, weighing balance. Procedure: Pour the powder by means of a funnel into a 100 ml graduated cylinder up to the mark and record this volume as Vo (100 ml). Mechanically tap the cylinder containing the sample by raising the cylinder and allowing it to drop under its own weight. Continue the tapping for 1000 times or until there is no further reduction in the volume. Determine the volume after tapping as Vf. Finally determine the Compressibility Index by the formula:( )
Vo = volume of the powder before tapping (100 ml). Vf = volume of the powder after tapping Compressibility Index as an indication of the Flow Property/ Classification of powders on the basis of Compressibility Index: Compressibility Index (%) 15 % 15-25 % > 25 % Type of Flow Free Flowing Intermediate Flowing Poor Flowing
Pencil Work foe Exp # 5:
Initial Volume (Vo) 100 ml 100 ml 100 ml 100 ml
Final Volume (Vf)
Type of Flow
NaCl Starch Kaolin Talc
EXPERIMENT#6 Title: To determine the Angle of Repose of the given Powders and to Characterize the given powders on the basis of Angle of Repose: Angle of Repose: The angle of repose is the minimum angle at which any piled-up bulky or loose material will stand without falling downhill. (or) The angle of repose or, more precisely, the critical angle of repose, of a granular material is the steepest angle of descent or dip of the slope relative to the horizontal plane when material on the slope face is on the verge of sliding. This angle is in the range 090.
Figure: Angle of Repose When bulk granular materials are poured onto a horizontal surface, a conical pile will form. The internal angle between the surface of the pile and the horizontal surface is known as the angle of repose and is related to the density, surface area and shapes of the particles, and the coefficient of friction of the material. Material with a low angle of repose forms flatter piles than material with a high angle of repose. Requirements: NaCl, Starch, Kaolin, Talc, Ruler. Procedure to Measure and Calculate the Angle of Repose: The powder is poured through a funnel to form a cone/pile. The tip of the funnel should be held close to the growing cone and slowly raised as the pile grows, to minimize the impact of falling particles. Stop pouring the material when the pile reaches a predetermined height or the base of a predetermined width (i-e when the powder falls of the edges of an inverted petri dish). Angle of Repose is then determined dividing the height of the cone by half the width of the base of the cone. The inverse tangent of this ratio is the angle of repose.
The equation for calculating the angle of repose is given as follow: tan = h/r tan = 2h/d .. = tan-1(h/r) (or)
.. = tan-1(2h/d)
where: = angle of repose h= height of the pile r= radius of the pile (distance from the centre of the pile to the edge of the pile) d= diameter of the pile Angle of Repose as an indication of the Flow Property/ Classification of powders on the basis of angle of repose: Angle of Repose (degrees) 25 25-50 > 50 Type of Flow Free Flowing Intermediate Flowing Poor Flowing
Pencil Work foe Exp # 6:
Height of the cone (h)
Radius of the cone (d)
Angle of Repose = tan-1(h/d) Type of Flow
NaCl Starch Kaolin Talc
Experiment # 7 Title: Preparation of medicated ophthalmic solution isotonic atropine sulfate solution (3.8% eye drops) Theory: Ophthalmic solutions are sterile products, must be free from particulate matter, intended to be instilled into eyes to have a desired effect. Osmosis: It is the net diffusion of solvent (water) from a region of high water concentration to a region of low water concentration through a semi permeable membrane. Osmotic pressure: The pressure required to prevent osmosis is called osmotic pressure. It is a colligative property. Tonicity: Each cell has its normal membrane tone called tonicity. It is measurement of effective osmolarity or osmolality. The tone of cell is measured in terms of NaCl, boric acid. Normal tone of RBCs is equal to 0/9% NaCl (W/V). For lacrimal fluids 1.9% boric acid solution.
There are three types of solutions. 1. Isotonic solution: A solution is said to be isotonic when there is no net gain or loss of water by cell or other changes in cell when it is in contact with that solution. 2. Hypotonic solution: A solution is said to be hypotonic when there is a net gain of water by cell when cell is inserted in that solution. 3. Hypertonic solution: A solution is said to be hypertonic when there is a net loss of water from the cell when cell is placed in that solution. Effect of isotonic solution: A cell when placed in isotonic solution will remain of same size & shape, as there is no net gain or loss of water by that cell. This is because a cell has the same tonicity as that of an isotonic solution. Example: A solution having osmolarity f 280mosm/L i.e 0.9% NaCl solution W/V, 5% glucose solution.
Effect of hypotonic solution: A cell when placed in hypotonic solution will swell. As concentration of water within cell is less. Example: A solution having osmolarity 280mosm/L. Equation: Amount of solute to make isotonic solution = Where: i; dissociation constant. Which depends on Nature of substance Extent of dissociation Number of ions present in molecules NaCl; extent of dissociation is 80%. NaCl dissociates into Na+ & Cl-. 2 x 80 = 160 ions & 20 are intact solute molecules, so net solute molecules are 180. So, i for NaCl is 180/100 =1.8 Now from equation 1 Amount of solute to make isotonic solution = Means that 0.9gm in 100 ml (0.9 %) x = 9.06g in 1000ml x . 1
NaCl equivalency value: We have to get NaCl equivalency value because as active is to be added, which also exert its own tonicity. Accordingly amount of NaCl is to be decreased. NaCl equivalency value = x
NaCl equivalency value = From equation 2 we get 0.12gm
= 0.12 gm
For each gm of atropine sulfare we have tyo replace 0.12gm of NaCl to achieve same tonicity.
Example: 3.8% atropine sulfate solution We know for 1gm , equivalent facter is 0.12gm. In 30 ml we have Amount of atropine sulfate is 0.3gm Amount of NaCl to be added is 0.15gm to get isotonic ophthalmic solution Requirements: Apparatus: Beakers, stirrers, balance. Chemicals: Distilled water NaCl Atropine sulfate
Procedure: For 3.8% atropine sulfate solution; 1. I took a beaker & placed in it accurately weighed 1.14 gm of atropine sulfate. 2. then I added to it 112 mg of NaCl.22
3. add small volume of distilled water, in order to dissolve them. 4. at end I make the volume to 30ml with distilled water to get isotonic atropine sulfate(3.8%) eye drops.
Calculations: For 100ml, We have to add 3.8gm of atropine sulfate For 30ml, 3.8/100 x 30= 1.14gm of atropine sulfate
NaCl to be replaced: 1gm atropine sulfate= 0.12gm of NaCl For 1.14gm atropine sulfate = 0.12/1 x 1.14 = 157.2mg or 0.1572gm
NaCl to be added: For 100ml = 0.9gm NaCl (isotonic solution) For 30ml= 0.9/100 x 30 For 30ml = 270mg of NaCl to be added to get isotonic solution Now amount of NaCl to be added= 270- 157.2 =112.8mg
Atropine sulfate (3.8%) eye drors (30ml) Atropine sulfate= 1.14gm NaCl= 0.1128gm Distilled water QS to make 30ml.