1. Areas where new development tools could accelerate progress -- Formulation -- Glycosylation

1. 1. Areas where new development tools could accelerate progress -- Formulation -- Glycosylation 2. Potentially important future areas of medical development -- Nanotechnology -- Tissue Engineering

2. Other variables process 1 solvent 2 3 4 Impact of TransForm Technology Traditional TransForm # of F & F experiments • 200-20,000+ • 10-20 Ability to explore F&F space more effectively and efficiently Timing • 2 – 4 Weeks • 1 – 2 Months Informatics; data mining, learning • Deep & iterative • Minimal

4. Case Study: Ritonavir • 1.5 years after launch, converted into unanticipated form II polymorph • 50% less soluble • Abbott compelled to recall & reformulate MPT 122 °C MPT 125 °C MPT 80 °C MPT 97 °C MPT 116 °C Form I Form II Form III Form IV Form V • Within weeks at TransForm, using < 2g: • Both known forms identified & characterized • Found three novel, previously unreported forms • Novel, robust methods to make each form Morissette et al. PNAS100, 2180 (2003).

5. New Tools • Imaging • Informatics • Genomics • Proteomics • 5. Glycomics

6. 3 B N R A G C T A G C - - C H O S O - C H O S O - C H O S O - O O C 2 3 2 3 2 3 3 O O O O O O O O - C O O - C O O O O O O O H O H O H O H O H O H O H O H O O - - - - - N S O - O S O N H S O - N H S O S O O O O O S 3 3 3 3 3 3 3 “Cracking the Code” of Sugars is Analogous to the Sequencing of DNA • The sequencing of DNA has laid the foundation for biotechnology revolution • Like DNA and proteins, sugars play a central role in regulating basic biological activity, disease mechanisms, and drug action • Sugars exist as sequences of building blocks similar to DNA, but there has been a lack of adequate sequencing tools • Understanding of sugars is critical for polysaccharide drugs (e.g. Lovenox) and glycosylated proteins (e.g. Epogen)

7. Inherent complexity of sugars has prevented comprehensive understanding • Structural complexity and information density • Lack of amplification • Heterogeneity The Problem: Lack of technology to and tools to sequence sugars has made it difficult to characterize and engineer sugars, and decipher their role in biology.

8. ESI-MS NMR Linkage information Mass signatures of groups MALDI -MS Mass of chain – chain length Quantitative building block information CE Multiple Enzymes Integration of Data Convergence on unique solution to complex sequences Sequencing Complex Polysaccharides [1999] Science 286: 537-542.Momenta Pharmaceuticals

9. Future areas of medical development 1. Nanotechnology 2. Tissue Engineering

10. Prototype Device Silicon Nitride or Dioxide Silicon Active Substance Cathode Anode

11. Implantable Drug Delivery System Battery-powered, telemetry-controlled implant Design based on pacemaker and ICD microelectronics

12. Reservoir Opening Mechanism

13. Pre-Clinical Studies Demonstrate in vivo Release • Experimental Protocol • Implant microchips subcutaneously in rats • Release radioactive mannitol (388 ng/well non-metabolized sugar) • Collect urine and analyze for radioactive content • as an indicator of drug release

14. hydrophilic block DNA cationic block polyplex polymer-DNA complex Polymer Therapeutics : Nanosized medicines polymer-protein conjugate polymeric drug or sequestrant protein Mw = 5 - 40,000 Da ~20nm 40-60 nm 5-15 nm targeting residue hydrophilic block drug linker hydrophiobic block drug polymer-drug conjugates polymeric micelle 60-100 nm

15. 1. How do you assess safety? • 2. How do you characterize nanomedicines • --Biological • --Physical/chemical • 3. What animal models are appropriate?

16. Annual Tissue LossEnd Stage Organ Failure (U.S.) • Over $500 billion in health care costs • 40 to 90 million hospital days • 8 million surgical procedures

17. Bone Joint replacement 558,000 Bone graft 275,000 Internal fixation 480,000 Facial reconstruction 30,000 Cartilage Patella 319,400 Meniscus 250,000 Arthritis (Knee) 149,900 Arthritis (Hip) 219,300 Small Joints 179,000 Tendon 33,000 Ligament 90,000 Skin Burns, Sores, 3,650,000 Ulcers 1,100,000 Heart 754,000 Blood Vessels 606,000 Liver 205,000 Pancreas 728,000 Incidence of Organ and Tissue Deficiencies

18. Biodegradable Polymer Scaffold In Vivo Implantation New Bone Cartilage Liver Intestine Ureter In vitro Tissue Culture Cells Osteoblasts Chondrocytes Hepatocytes Enterocytes Urothelial Cells

20. Cartilage Tissue Engineering BEFORE cell seeding AFTER 2 weeks in culture

22. System • Modified PGA Tubes • 8 Weeks SMC Culture, then EC • Bio-Reactors – Pulsatile Radial Stress

23. MediumReservoir Flow Direction 4 Bioreactors, Assembled in parallel 20 cm Pulsatile Pump Compliance Chamber Magnetic Stirplate

25. Characteristics • 50% Collagen • Rupture Strengths > 2000 mg Hg • Suture Retention – Strengths up to 90g • Demonstrates Contractile Responses to Serotonin, endothelin-1, and Prostaglandin F2α

27. Human Embryonic Endothelial Cells Form Functional Blood-Carrying Microvessels PECAM1 CD34

30. How should safety be assessed? • What are appropriate markers? • How do you determine appropriate function? • What are appropriate animal models?