A Formula for Success

1. A Formula for Success

2. Scenario 1: • You are a medicinal chemist for a small biopharmaceutical company. One of the drugs that they are interested in developing is a lysozyme tablet that can be taken to help fight certain bacterial infections. Your job is to develop a formula for the excipients in the tablet. Where do you begin?

3. Excipients • Inactive substance of a drug that holds the active ingredients. • Holds drug together (binder) • Coatings –addresses taste and size • Preserving – extends expiration date • Dilution of dosage/fillers • Stability/buffers • Lysozyme – enzyme that disrupts cell wall of bacteria, found saliva, tears,

4. Scenario 2: As part of the development team, you want to focus your formula to address pH, taste, and fillers. The plan has now changed to formulate this for capsules. You have not received the active ingredient data yet so you must move on in your development of the formula. What ingredients will you use and at what concentrations?

5. Buffer Design • Design an experiment to help you decide the best buffer and concentration to use. This may require multiple experiments. • Remember to: • Decide on buffer and concentration first • Do the calculations to help you weigh right amount • Use distilled water and add less water than needed to give you room to add acid or base as you adjust pH (near 7) • Test buffer efficacy by adding acid in increments and checking pH

6. Other components • Taste – sugars (sucrose, dextrose, fructose) • Which one? Mass/volume” • Fillers – starch or gelatin • Which one? %?

7. Scenario 3: The folks working with the lysozyme have been pulled over to into a new project and now you have been given the job to determine what is the best concentration to use in the proposed drug. You will need to design an experiment to determine this.

8. Making Media Day 1 • Make up 100 ml of Nutrient Agar and 50 ml of Nutrient Broth. Each can go in a separate, labeled 100 ml media bottle. Add distilled water to correct volume. • Autoclave to kill all living organisms. Day 2 • Melt agar and pour plates • Inoculate plates with bacteria and incubate overnight Day 3 • Pick colonies and inoculate 25 ml of Nutrient Broth in a sterile culture tube and incubate overnight.

9. Sterile Technique • Follow SOP for Sterile Technique in Lab Manual • Pour a starter plate and allow to solidify • Triple Z streak on your plate to inoculate it and incubate overnight • Transfer 20 ml of Nutrient Broth into sterile tube • Pick colony and inoculate broth. Incubate overnigth

10. Lysozyme Assay • Make enzymes up in desired concentrations in buffer. Concentrations of 1-10 mg/ml work well. • Set Spec at 450nm • Blank spec 20 with buffer • Pour 3 ml of bacteria culture into each tube. Check to see the OD450 is around 0.8. If not, add buffer to bring it down to that range. • After recording the absorbance, add 0.1 ml of enzyme to tube and monitor. • Repeat for other enzyme concent.

11. Scenario 4: It has been discovered that the acid concentration of the stomach has a major impact on the activity of the active ingredient. You now need to determine the concentration of the acid and then adjust the buffer to improve the maximum activity of the lysozyme

12. Titration to Determine Acid Molarity • Pour 25 ml of the “stomach acid” into a beaker. • Add two drops of phenolphthalein to the solution • Pipet 1 ml of 1M NaOH using a 1ml pipet. • Add the NaOH to the beaker one drop at a time while swirling the beaker. • You should see the solution turn pink for a second and then disappear. • As you get near to the end point (neutralization), it will take longer for the pink to disappear. • At some point, the pink will not go away as you have passed neutral and the solution is now basic. • Record the total amount of NaOH needed to reach the end point . • To calculate the molarity of the solution use the following equation: • MA x VA = MB x VB • where M is molarity, V is volume, A is acid (HCl in this case) and B is base (NaOH in this case)

13. Scenario 5 • The senior staff likes the data you have generated so far and wants to move forward with the development of the drug. However, marketing is asking that the drug be given a distinguishable color. They are concerned however about the dyes that are used due to adverse reactions. You need to research the dyes and find a single, negatively charged dye to use.

14. Scenario 6: You and your team have completed our formulations for the new drug. You are to submit at least two capsules to quality control for testing. How close you are to meeting project goals determines how large of a bonus your team gets. Your team may also get to name the new product.