Vikram Ramanujam 1
This proposal discusses the necessity for the production of a fast acting insulin analog in order to improve the quality of life of diabetes patients. Fast acting yet stable insulin is necessary to produce effective insulin pumps that do not have oscillations. Insulin pumps are already in wide scale use, but users currently run the risk of keto-acdiosis if there is a disconnection and a host of other illnesses as a result of oscillations. A literature review is included to show the impacts diabetes has on quality of life, the modern advances in insulin pump technology, and the role that a fast acting insulin analog would play in further improvement of these pumps. This proposal outlines a thorough procedure for determining how to find the most suitable analog for producing fast acting insulin. This includes using a program called CHARMM to conduct Quantum
Mechanics and Molecular Mechanics (QM/MM) calculations with various halogen substitutions at different positions of the benzene ring in the phenylalanine-24 amino acid of the beta chain of insulin. Iodine, Fluorine, and Chlorine are the planned substitutions outlined in this proposal.
This proposal is directed towards DDRI, an organization in the city of Cleveland that supports student diabetes research. No materials need to be obtained for this project because it is all available in the lab of the principle investigator, Dr. Michael Weiss. DDRI is a company focused around diabetes research and hence it is imperative that this research be conducted so that a breakthrough might be achieved in improving the quality of life of diabetes patients. Producing a successful fast acting insulin analog will aid in the creation of useful and stable insulin pumps and will also help quell the problems that occur if a pump were to be disconnected. These advances would propel diabetes treatments to a new level.
My proposed research project involves a new approach to dealing with Diabetes. The aim is to develop an ultra-fast yet stable insulin analog to enhance the therapy of type 1 diabetes mellitus.
This stable ultra-fast insulin analog will allow for the production of non-oscillating insulin pumps. Advances in the engineering of continuous glucose monitors and “smart” insulin pumps in principle will enable design of automated “closed loop” systems (i.e., an “artificial b-cell”).
The necessity for this research stems from the fact that the current formulations of insulin are not very fast acting (even with the “meal-time” substitutions introduced in the 1990s by Eli Lilly and
Novo-Nordisk to create Humalog and NovoLog, respectively). My work at CWRU may thus lead to a breakthrough in the design of fast-acting insulin analogs.
This may in turn enable key clinical applications in the future. “Smart” insulin pumps can be used with ultra-fast insulin and feedback algorithms can be designed to control these pumps. An ultra-fast insulin analog formulation is necessary because the current insulin formulations are inadequate in that delayed absorption interferes with the safety and effectiveness of the control

Vikram Ramanujam 2 algorithms used in closed-loop systems. For example, instability in the current feedback algorithms can cause oscillation in insulin values and in blood glucose concentrations.
The purpose of this literature review is to discuss the existing studies on insulin pump technology and discern the role that fast acting insulin analogs might have on improving this technology. The literature review will focus on the advantages of insulin pump technology versus traditional injection methods, and will elaborate on the current problems associated with this technology. By necessity, the problems and diseases associate with diabetes will also be discussed.
Introduction
Because it causes a glucose imbalance that must constantly be monitored, diabetes is a disease that greatly diminishes the quality of life of its victims. If left unmonitored for even a short time it can cause severe bodily harm and even death. Published studies show that diabetes sufferers have abnormal blood sugar levels that cause physiological and psychosocial problems. Studies also indicate that insulin is a great product that can be used to control blood sugar abnormalities and help improve patient quality of life. What studies disagree on, however, is the most efficient and effective means of administering insulin in order to control diabetes. While diabetes is incurable, technological progress has created several useful methods of controlling it. The primary method used to control diabetes is through various applications of insulin and insulin analogs. Several ideas and studies have been proposed with the most recent research developments regarding insulin pumps. In order to understand and justify the necessity for insulin analog-dispensing smart insulin pumps, it is essential to look at various published works in order to understand the various diseases that diabetes presents, the nature and function of insulin pumps, and the effects that ultra-fast insulin would have on currently available insulin pumps.
Quality of Life Deprecation
Diabetes presents several new found detriments to quality of life. Recent studies have shown that people with diabetes are now prone to developing psychiatric disorders, eating disorders, and developing substance abuse problems (
Bourdel-Marchasson, Fagot-Campagna, & Helmer, 2009). These problems lead to further complications beyond just simple hypoglycemia and hence result in a greater burden on the medical industry to treat these patients. In addition, diabetes patients, especially older ones, are now being more prone to develop functional limitation and activity restriction in daily life activities (Bourdel-Marchasson et al., 2009). Lack of blood sugar regulation already is life threatening if untreated, but now research has presented startling evidence about further perils associated with diabetes and the need to control it as much as possible.
Insulin Pump Technology

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Insulin pump technology is a relatively new innovation in administering insulin to diabetic patients. The technology works by allowing the patient to wear a small device that has a reservoir of insulin. The device has a needle that can be placed under the skin and the pump administers insulin to the patient at varying amounts throughout the day. This system is capable of replacing the traditional three to four a day insulin injections that many patients are accustomed to using (Grant, 2007; Renard et al., 2009; Ibini, 2007). This revolutionary system of pumping insulin into patients seems to be the best method of insulin delivery. The advantages of this pumping system include a system of control for blood sugar, allowing for a lot of the diseases associated with diabetes to be contained. In addition, the patient does not have to worry about facing life threatening circumstances if they miss one injection during the course of a day, making pumps a valuable commodity. While insulin pumps seem flawless, the reason for continued research presents itself because insulin pumps have some tremendous downsides. Any problems with the pump or disconnections can cause grave health issues. Since there is only a short supply of insulin in the pump, these types of technical issues will typically lead to rapid hyperglycemia and hence result in diabetic keto-acdiosis. Essentially this means the blood pH will drop and the blood becomes acidic which leads to death (Grant, 2007; Weiss & Phillips,
2008).
Adaptive controls have been developed to attempt to mitigate this keto-acdiosis. None, however, have yet to be successful. One of the methods developed is to have fuzzy logic control. What this does is discern when the patient needs more insulin and when they need less based on biological sensory detectors (Grant, 2007; Renard et al., 2009). However, the problem with this control system is that it produces oscillations. The body is unpredictable and hence it is hard to pinpoint the exact amount of insulin needed at all times direct without human involvement. Since the goal is to produce self-regulating pumps to allow patients to be free and not have to worry, producing non-oscillating levels of insulin without human involvement is the new goal in industry (Renard et al., 2009; Ibini, 2007). It is essential to prevent this oscillation cycle because, while this minimizes keto-acdiosis, other issues may arise such as alkalosis. This condition occurs when the blood pH level is too high and hence the blood becomes too basic. Essentially this will produce the same end result (of death) that acidosis causes (Grant, 2007; Weiss & Phillips, 2008).
Fast Acting Insulin
This is why there is a need for fast acting insulin analogs. The goal of a fast acting insulin analog is to supply the necessary insulin needed to control blood sugar at super fast rates. This gives a twofold benefit. First, fast acting insulin is necessary to produce “Smart” insulin pumps that use feedback algorithms to control these pumps. An ultra-fast insulin analog formulation is necessary because the current insulin formulations are inadequate in that they cause delayed absorption, which interferes with the safety and effectiveness of the control algorithms used in closed-loop systems. Using fast acting insulin, oscillation cycles can be prevented because there will be rapid feedback to determine how much insulin needs to be pumped into the patient. Hence this will prevent the issues such as alkalosis that are seen in modern day pumps because of a lack of oscillation. This allows for practical use of an “artificial beta-cell” (a non-human controlled self sufficient insulin pump) that in turn can improve glycemic control with lower risk of both hypoglycemia and hyperglycemia. Second, in case there is a disconnection in the insulin pumps, fast acting insulin analogs can prevent keto-acdiosis. If the pump breaks, a patient can still take a

Vikram Ramanujam 4 normal injection using a regular needle. However, normally, after a certain amount of time the body will go into keto-acdiosis and hence taking a normal shot will likely not help. Nevertheless, with fast acting insulin, the insulin will react fast enough that even if a lot of time passes, the keto-acdiosis can be prevented (Weiss & Phillips, 2008).
Conclusion
Throughout the last few decades, major advancements have occurred in the medical industry to determine the broad reaching effects that diabetes can have on the quality of life of patients. It has been determined that the effects are not just physical but extend also to mental disorders including eating disorders, drug use, and psychiatric problems. Many researchers have conducted experiments to determine that insulin is useful in quelling and controlling diabetes despite the fact that it is incurable. The use of insulin pumps are a new innovation that allows for systematic injection of insulin into the body without the patient having to worry about multiple daily injections that present life threatening circumstances if missed. However, this insulin pump system is still dangerous and breaks in the pump system can lead to keto-acdiosis which is itself a life threatening disease. Further industry developments made to improve upon this system still produce oscillations, which, while cut down on keto-acdiosis percentage present other equally fatal conditions. Creating fast acting insulin pumps will do many things. First it will prevent the oscillations that are seen in traditional pumps that exist today. It will also allow for injections using normal needles to have a much greater chance to prevent keto-acdiosis since the insulin will act a lot faster. This is why creating fast acting insulin analogs to replace traditional insulin in pumps can help create safe and usable insulin pumps that will dramatically help improve the quality of life of many diabetic patients and lead to a revolutionary breakthrough in the field of medicine.
From September of 2009 to right now I have been working in Dr. Michael Weiss’ research lab.
He is the chair of the Biochemistry department at Case Western Reserve University and is working on the fast acting insulin project that I have discussed. I have been currently involved working in his research lab dealing with a similar project that looks to produce a long lasting insulin analog. During this project I have learned a lot of the QM/MM techniques needed in insulin analysis and have become very familiar with the specifics of Dr. Weiss’ lab and the various people working there. I have the skills needed to perform simple tests such as Western
Blots, Gel Electrophoresis, and Pippetting that are required to be successful in the lab. I have been working with biology and chemistry concepts since middle skill and my coursework at
Case Western Reserve in Biology, Chemistry, and Organic Chemistry has allowed me to gain a strong understanding of biological and molecular design. My academic success coupled with the months I have spent working on a very similar research project ensures that I have the skills necessary to successfully perform this project.
In order to determine the best insulin analog to produce, one must first observe carefully the complex structure of Insulin. Below is a diagram of the tertiary structure of insulin:

Vikram Ramanujam 5
Insulin Structure Diagram+
As this diagram shows, an insulin monomer is made up of two principle chains (the alpha chain in grey and the beta chain in black). The diagram on the right also shows where the C (starting) and N (ending) terminus is in each chain.
The method for producing fast acting insulin analogs relies on analyzing this structure and then making changes to it. This structure is complicated, as the above diagram shows. Not only are there multiple chains, but they intertwine and interlace with one another. This means that slight changes at any point in the structure will have significant impacts on the structure as a whole because the nature of the bonding will be adversely affected. The principle idea for making fast acting insulin focuses on phenylalanine, the 24 th
Amino Acid in B-chain. This amino acid is primarily composed of a benzene ring, which is composed of six interconnected carbon atoms joined in a ring shape with two hydrogen atoms linked to each Carbon atom. This structure is detailed below.
Benzene ring structure+
The idea behind making fast acting insulin is that by substituting the hydrogen atoms in the ring with halogens, the chemical properties of the amino acid will change. As a result of the complex bonding patterns found in insulin this slight change will transform insulin as a whole to an unpredictably acting analog.

Vikram Ramanujam 6
However, a method has been developed to determine the theoretical effects that making changes to the beta chain will have on molecular stability. This method is known as QM/MM which stands for quantum mechanics/molecular mechanics. The math used to derive these effects is higher level quantum theory. This is difficult math to understand, but fortunately a computer program called CHARMM was designed at Harvard that does the calculations in order to determine the thermodynamic stability, provided that accurate and useful data is given as inputs.
The program is a biomolecular simulation program that is highly versatile; it focuses on peptides, lipids, nucleic acids, and small molecule ligands and their behavior in various environments.
Using CHARMM and employing the QM/MM calculations, theoretical data about the most useful and stable substitutions can be collected through multiple simulations. The current plan is to test Iodine, Fluorine, and Chlorine at positions 2, 3, and 4 in the benzene ring and determine which substitution is capable of giving the best thermodynamic stability. Depending on the nature of the results, other halogenations might be tested.
After running theoretical tests, the next goal will be to verify them using experimental tests. In the lab, the different insulin analogs deemed to be the most theoretically stable will be synthesized and employed in actual insulin pumps to test for how fast acting they are. Upon having both experimental and theoretical data it will be possible to quantify what is the best analog to use as the fast acting insulin. The goal for future projects will then be to make use of this newly developed fast acting insulin to effectively and efficiently improve and control insulin pumps.
Below is a schedule for a the activities that need to be performed during the course of the research project
QM/MM Calculations – Introduction and Fluorine
Week 1: Learn how to use the CHARMM program and construct models of insulin based on experimental structure
Week 2:
Determine molecular stability of regular insulin
Determine molecular stability of analog with 2-Fluorine
Determine molecular stability of analog with 3-Fluorine
Determine molecular stability of analog with 4-Fluorine
Week 3: Determine molecular stability of analog with 2,3,4-Fluorine
Determine molecular stability of analog with 2,3-Fluorine
Determine molecular stability of analog with 3,4-Fluorine
QM/MM Calculations –Chlorine Calculations
Week 4: Present conclusion on the best acting Fluorine Insulin analog

Week 5:
Week 6:
Determine molecular stability of analog with 2-Chlorine
Determine molecular stability of analog with 3- Chlorine
Determine molecular stability of analog with 4- Chlorine
Determine molecular stability of analog with 2,3,4- Chlorine
Determine molecular stability of analog with 2,3- Chlorine
Determine molecular stability of analog with 3,4- Chlorine
QM/MM Calculations –Iodine Calculations
Week 7: Present conclusion on the best acting Chlorine Insulin analog
Week 8:
Determine molecular stability of analog with 2- Iodine
Determine molecular stability of analog with 3- Iodine
Week 9:
Determine molecular stability of analog with 4- Iodine
Determine molecular stability of analog with 2,3,4- Iodine
Vikram Ramanujam 7
Determine molecular stability of analog with 2,3- Iodine
Determine molecular stability of analog with 3,4- Iodine
Experimental Calculations
Week 10: Present conclusion on best acting Iodine insulin analog
Determine best over-all Insulin analog
Week 11: Perform experimental synthesis using the compounds found to be the most theoretically stable and then test for empirical stability
The majority of the budget for this project will be covered by the department of Biochemistry.
All insulin compounds, test software, materials, and lab ware will be available at for use in Dr.
Weiss’ research lab. The estimated budget represents the desire for a stipend. This stipend assumes campus minimum wage payment for the duration of the project.

Hours Worked/Week Wage Rate
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Weeks Worked for
Summer
Estimated Total
Stipend
[Product of first three columns:
($9.25/hr)*(11 wk)
*(40 hr/wk)]
40 Hours/Week $9.25/Hr 11 Weeks $4070
For this project, a lot of collaboration will be required. The goal of the project is to find the best fast acting yet stable insulin analog and hence other students working in the lab will also be running QM/MM calculation models with different analogs. At the end of the summer all of the students working here will compare results and select the best analog from the research findings.
Dr. Weiss will be guiding us through our research and I will be specifically assigned to work under post-doc Dr. Nalinda Wickramsingh.
______________________________________________________________________________
+ The images used in this proposal were created by author, Dr. Michael Weiss
Bourdel-Marchasson, I., Fagot-Campagna, A., & Helmer, C. (2009). Disability and quality of life in elderly people with diabetes. Diabetes and Metabolism , 1 (33), S66-S74.
Grant, P. (2007). A new approach to diabetic control: Fuzzy logic and insulin pump technology.
Medical Engineering & Physics , 29 (7), 824-827.
Ibbini, M. S. (2009). Comparative study of different control techniques for the regulation of blood glucose level in diabetic patients.
33 (8), 656-662.
Journal of Medical Engineering & Technology ,
.(2009). Psychosocial problems in adolescents with type 1 diabetes mellitus.
339-350.
Diabetes & Metabolism , 35
Renard, E., Meyer, L., Melki, V., Fermon, C., Schaepelynck-Belicar, P. et al. (2007). Ten
(5), years of experience confirm the favourable effects of insulin pumps implanted in badly diagnosed type 1 diabetes. Diabetes & Metabolism , 33 (Sp. Iss. 1), S66.
Weiss, M., & Phillips, N. (2008). Design of an Active Ultrastable Single-chain Insulin Analog
Synthesis, Structure, And Therapeutic Implications. The Journal of Biological Chemistry ,
283 (21), 14703–14716.