This project is dedicated to the task of identifying the molecular structure of an inhibitor to chemically shield the significant components of damaged cartilage resulting from osteoarthritis. This shielding will result in selectively blocking the degeneration of chondroiten sulfate, the main constituent of cartilage matrix whose degradation is largely responsible for the softening and deterioration of patellar cartilage. The degeneration of chondroiten sulfate, whose structure is close to that of Hyaluronic acid, is precipitated by the enzymatic activation of the enzyme, Hyaluronidase, that reduces the concentration of Hyaluronic acid in the synovial fluid in order to stabilize its pH at between 7.2 and 7.4.
The main thrust of this project is to research and develop a marketable biochemical product that will shield chondroiten sulfate from enzymatic degradation and thereby possibly halt or reverse symptomatic osteoarthritis resulting from this degradation of cartilage. Once that product is identified and determined, Siskiyou Products will sub-contract or sell to one or more pharmaceutical companies the rights to market the finished Commercial Prototype to the general public for profit.KEYWORDS: Cartilage; Chondroiten Sulfate; Hyaluronidase; Hyaluronic Acid; Inhibitor; Osteoarthritis, Chondromalacia Patella
Part 0: Legend and Definitions
HA: Hyaluronic Acid Co: Collagen
CS: Chondroiten Sulfate OA: Osteoarthritis
HAase: Hyaluronidase CP: Chondromalacia Patella
Inhibitor: The molecule required to inhibit the action of HAase against CS by forming temporary bond with CS, without affecting HAase itself. This molecule is referred to as Inhibitor and further defined in §3.7.
Intermediate: The molecular species formed by the temporary bond between an Inhibitor and CS from the electrochemical affinity between the inhibitor and the SO4 constituent of CS.
Commercial Prototype: The Inhibitor that exists at the successful completion of Stage 4 as described in §3.2.4.
Proprietary Information: The only portion of this proposal that is Proprietary Information is the Research Plan, which is Part 4 of this Project Description. The declaration of Proprietary Information is designated under the Part 4 Research Plan title with the font shown with this sentence.
Part 1: Identification and Significance of the Innovation
1.1 The fundamental goal of this Project is to regenerate degraded cartilage tissue by a new medicine engineered by an innovative biomedical technology whereby the electron configuration differences between HA and CS are considered in order to shield the CS substrate from degradation rather than deactivate the degrading enzyme. As a result of this technology, the rate of tissue regeneration is expected to be greater than the rate of tissue degradation, thus resulting in a net gain of regenerated tissue. For a discussion on the broader impacts of this proposed Project, please see §4.9.
OA is a complex disease in which tissues of the cartilage, synovial membrane, and subchondral bone play significant roles21. Even though articular carriage degradation is a major characteristic of OA, it is still not completely understood what initiates its degradation and loss in all cases. Synovial membrane inflammation is believed to play an important role in the progression of joint tissue lesions; however, synovial inflammation is a secondary rather than primary contributor to the progression of OA. It is known that Outerbridge’s Ridge is one etiology. It is also known that major traumas and normal wear and tear are etiological factors as well, especially in older patients. The underlying hypothesis behind this Project is based upon the vicious cycle described in §2.2, thought to occur due to trauma or normal wear and tear. It is also assumed that the many degenerative changes within the matrix of articular cartilage are closely associated with the decomposition of CS, and therefore, if the hydrolysis of CS can be halted, so too can the degeneration of articular cartilage20. This Project will be concerned with undertaking the task of selectively shielding damaged cartilage at the molecular level in the lesions of CP, OA, or, as they are collectively known, degenerative joint disease. This chemical shielding is discussed in detail in §1.3.
1.2 HAase appears to be the most effective enzyme that is successful in hydrolyzing CS and HA, although other enzymes that are present, such as hexosaminidase and suftatase may be effective as well. Stages 1 and 2 of this Project, described in §3.2, will comprise Phase I where HAase will be primarily used to test the effectiveness of the Inhibitor introduced to form a protective Intermediate with CS against degradation. Those Inhibitors that are found to be successful in forming a temporary bond with CS and thereby stabilizing CS against HAase will be tested in like manner against the other enzymes commonly present in OA during Stage 3 of this Project to ensure CS stability.
1.3 Innovative Aspect: There is nothing new about deactivating enzymes in order to halt the biochemical processes in which they are involved. In fact, it has been standard operating procedure for decades in the field of biochemistry to inhibit enzymes from catabolizing their targeted substrates in order to preserve the integrity of those substrates from degradation. In those cases where a particular enzyme is hyperactive and/or behaving abnormally, this seems to be a wise approach with which we have no disagreement. However, when an enzyme is chemically behaving in a manner that is consistent with normal levels of activity but it is non-selectively degrading substrates that should not be degraded, then we believe inhibiting that enzyme is a gross mistake. What is innovative about this proposal is that instead of deactivating the enzyme, we will instead restrict its activity away from those substrates we understand should not be degraded, and allow the normal function of the enzyme to catabolize its intended substrate.
In those investigations where hyaluronidase or hexosaminidase inhibitors have been introduced into the synovial fluid, even where results have many times seen a temporary reduction in osteoarthritic symptoms, the procedures have resulted in an accumulation of HA in the joint, which causes new problems of its own because while symptomatic OA is sometimes alleviated, the increased acidity and concentration of HA begins to stimulate chondrocytes to secrete more HAase, continuing the cycle described in §2.2. In contrast, we will be indirectly “deactivating” enzymes on selected molecular substrates (i.e., CS) without actually affecting the activity of the enzyme itself. This approach will allow the enzyme to degrade its intended target and should eliminate the malfunction of the system as a whole, thereby greatly decreasing undesirable side effects associated with the non-selective inhibition of enzymes.
To summarize, the innovative aspect of this proposal is concerned with creating a loose and temporary bond from the electrochemical affinity between an inhibitor and a part of the targeted substrate under specific conditions, while not affecting the activity of the enzyme itself. This temporary bond is referred to as an Intermediate in §2.1E. In accomplishing this, the CS within the matrix of cartilage will be shielded from osteoarthritic degradation processes, while degradation of the unwanted HA within the synovial fluid will be allowed. Furthermore, when the pH of the synovial fluid stabilizes at normal values again, the Intermediate formed between the Inhibitor and CS will dissociate allowing the now inert Inhibitor to ineffectively remain within the synovial fluid or be removed through the synovial membrane because of its small molecular size. This will result in maintaining cartilage integrity; it will be the main objective of Stage 4 (described in §3.2) of this Project to analyze whether this cartilage integrity is enough to halt or reverse the effects of OA.
1.4 To accomplish the selective inhibition of HAase, or to be more precise, the specific shielding of CS during HAase activity, we will be considering molecular size, pH of the environment, electrochemical affinity of the SO4 constituent of the substrate to the inhibitor, molar concentration of the various substances that are appropriate to be considered, and even the viscosity of the synovial fluid itself. In all but the most specific chemical conditions, the molecule introduced to form a protective Intermediate with CS needs to be totally inert in order to avoid any side effects whatsoever. To handle these and other parameters, a new software program may need to be developed to sift through the millions of molecular combinations in order to match a unique molecular structure to the specific requirements for the HAase inhibitor we are seeking and, at the same time, that will guarantee inert behavior in all but the specified conditions for the specified target substrates, which are discussed in detail in §3.7.
1.5 High Risk: Non-intrusive medical solutions for the biochemical reversal of degenerative joint disease are currently non-existent12. There have been many investigations over the last 4 decades to halt or reverse degenerative joint disease, but all without success.13 Medical and biochemical journals are filled with such studies and investigations, most of which are concerned with an isolated aspect of the biochemistry involved in degenerative joint disease. To suggest a procedure that introduces an inhibitor that selectively inhibits HAase, not by deactivating that enzyme, but by chemically shielding a single substrate from degradation by the active HAase because of its electrochemical affinity for a constituent within the molecules of CS under highly restrictive conditions is a major deviation from orthodox biochemistry. If successful, then this shielding will also maintain the chemical integrity of CS not only when in the presence HAase, but when in the presence of other enzymes as well, such as hexosaminidase and glycosidase. But unfortunately this deviation, although innovative, does not guarantee technological success.
The collective biochemical interactions within the synovial capsule in both normal and degenerative cartilage are staggering. The idea behind this proposal is to avoid interfering with much of that biochemistry, and concentrate our efforts to those species that are being catabolized and resulting in deteriorated cartilage. If the activity of HAase (among others) on CS is largely responsible for degenerative changes in articular cartilage as well as secondary inflammatory changes of the synovial membrane, but HAase is necessary for the removal of HA, and the other enzymes present are required for the normal breakdown of other substrates, then the basic premise of this project questions the wisdom of deactivating any of the enzymes present. Instead of interrupting the entire articular system with unknown consequences and side effects by deactivating any given enzyme, let us instead block the one degradation in which we are interested: that of CS within the matrix of articular cartilage. This may be a simple and logical thought, as well as an effective approach. But exactly how do we accomplish that without adversely affecting the general biochemistry within the synovial capsule by the introduction of another chemical species that shields CS from degradation? That is the subject of this proposal: to determine, identify, and introduce an inhibitor that does not affect the activity of HAase or any other enzyme and, at the same time, is active in bonding with CS under very restrictive conditions in order to shield CS from degradation, but inert under all other conditions. It may be that this concept and this approach will not succeed, in which case this investigation will join the many others that failed to find a non-intrusive biochemical solution to degenerative joint disease. But if success is in fact attained with this Project then not only are the social implications far reaching, but the commercial implications are very promising as well, as discussed in Part 4 of this Project Description, “Commercial Potential.”
Part 2: Background and Phase I Technical Objectives
PHASE I: TECHNICAL OBJECTIVES:
2.0 Before any molecule is determined to accomplish the selective inhibition of HAase on CS, while not inhibiting its action on HA and ensuring its inert properties on the system as a whole, it is imperative that the minimum and optimum chemical and physical conditions affecting HAase activation and deactivation, as well as the synthesis and degradation of HA and CS, be investigated, defined, and documented. SBIR Phase I will be concerned with accomplishing these definitions and parameters. We will need to know the minimum and maximum conditions such as pH, rates of hydrolysis at each pH level, rates of synthesis and introduction of chemical species, variations in concentration as well as other, yet undefined, parameters (such as synovial capsule porosity and synovial fluid viscosity, which would set limits on molecular size of the inhibitor) that will become important as the investigation progresses.
2.1 Technical Objectives. The Primary objective of the Phase I effort is to successfully identify the Inhibitors (A-D below) capable of forming an Intermediate with CS, which will later become the Commercial Prototype(s) used for bringing the product to market. Without this identification, Stages 2-4 described in §3.2 are impossible to accomplish. When all of the Technical Objectives of this Project are reached, Siskiyou Products will have produced a finished product, referred to as the Commercial Prototype, that will be presented to one or more pharmaceutical companies for the purpose of being marketed and commercialized to the American public. This Commercial Prototype will not simply alleviate pain and symptomatic OA of those who purchase and use the finished product, but will halt or reverse the degradation of articular cartilage. These Technical Objectives are as follows:
A. To define the Inhibitor(s) that allow maximum hydrolysis of HA by HAase at high pH values.
B. To define the Inhibitor(s) to be effective in not allowing hydrolysis of CS by HAase at any pH value.
C. To identify the Inhibitor(s) concentration not affected by the presence or activity of HAase.
D. To ensure the Inhibitor(s) have no effect on the concentration or activity of HAase with all four species present, i.e., CS, HA, HAase, and the Inhibitor. In addition, A, B, and C above must be true with all four species present.
E. To identify the mechanism by which the CS-Inhibitor intermediate is formed.
F. To ensure the Inhibitor(s) are of a molecular size that is within the limits of synovial membrane porosity.
G. To ensure the Inhibitor(s) are inert in all appropriate physiological environments and non-reactive with normal cells and normal chemicals present in blood, urine, and synovial fluid.
H. To find an Inhibitors(s) that is not susceptible to uptake by normal tissues present with which they may come into contact. These tissues would include veins and arteries, the synovial capsule, fatty tissue, the bladder, kidney, and other tissues that the Inhibitor might encounter.
I. To determine the delivery system by which the Inhibitor will be introduced into the synovial capsule.
J. To determine the extent of cartilage degradation that can exist before treatment with the Inhibitor will have little or no effect.
PHASE I: BACKGROUND:
2.2 The Seven Steps outlined below are described here as a background to the overall events that occur in Degenerative Joint Disease. This Project, including SBIR Phase I, is concerned with the reactions occurring in Step 4.
The Seven Steps shown below describe a relatively simplified natural sequence of events that cause the molecular degradation of cartilage, where trauma or normal wear and tear can be shown to be the etiology of the lesion. Please note that these are not the only reactions taking place in osteoarthritic joints, for the process is a multifactorial process involving a number of enzymes2. But it is nevertheless the cyclic nature of this sequence of events with which this business endeavor is concerned because our goal is to maintain the chemical integrity of CS against any of the species present.
Step 1: Rise in pH The normal pH of synovial fluid ranges between 7.2 and 7.4. When a physical trauma occurs at the knee joint, whether the trauma is major or minor in nature, loose mucin cellular material is introduced into the synovial fluid from the internal walls of the synovial capsule. This results in an increase of pH until it is clearly basic in nature.
Step 2: Secretion of Hyaluronic Acid When the pH of synovial fluid reaches a significant level of alkalinity, HA is then secreted by chondrocytes that begins to lower the pH to acceptable levels1. If the HA is allowed to remain in the synovial fluid indefinitely, then the pH will become acidic, below the normal values of 7.2-7.4.
Step 3: Activation of Hyaluronidase When the pH of synovial fluid reaches an acidic threshold, HAase is activated to hydrolyze the HA, thus stabilizing the pH at acceptable values of neutrality. Accumulation of HA in the joint could result in the degradation of the joint by stimulating chondrocytes to secrete more HAase, and must therefore be avoided.
Step 4: Hydrolysis of Chondroiten Sulfate CS, one of the main constituents of cartilage matrix, and responsible for its solidity, is very similar in molecular structure to HA. The essential difference between the two molecules is the presence of the sulfate constituent within the former. And because HAase is a non-specific enzyme, the CS that is exposed to the synovial fluid is partially hydrolyzed along with the HA existing within the fluid. This is the critical step that this business endeavor seeks to block with a marketable chemical inhibitor. Blocking this step via the active site of the CS molecule (the SO4 constituent) will essentially end the molecular degradation of cartilage matrix and allow the rate of regeneration of cartilage to surpass that of degradation. Currently, cartilage does not regenerate because the rate of enzymatic degradation of CS greatly surpasses the rate of CS synthesis. This business endeavor is concerned with reversing those two rates.
Step 5: Resumption of normal pH within the synovial fluid As the HA is degraded by HAase, the pH of synovial fluid gradually increases to normality (7.2-7.4), at which time, Hyaluronidase is deactivated and both HA and CS are no longer degraded or hydrolyzed.
Step 6: Rise in pH due to SO4= As a result of the hydrolysis of CS in Step 4, there will now exist traces of SO4= radicals within a neutral synovial fluid. This SO4= will act as a weak base and increase the pH once again by its mere presence. If this business endeavor is accepted and funded by SBIR, then this SO4= will not exist in great quantities because Step 4 above will have been blocked. Therefore, the pH will not rise again and the cyclic nature of CS degradation, i.e., cartilage matrix, will have stopped at the molecular level.
Step 7: Degenerative Joint Disease If the rise in pH from Step 6 reaches the level described in Step 2, then the cycle repeats itself and the cartilage surrounded by synovial fluid is gradually degraded by the minute changes in CS. Alternatively, upon repeated traumas to the knee joint as time goes on, the same process as described above will continue to degrade the cartilage until the effects of CS degradation manifest themselves as pain, joint discomfort, and OA.
Part 3: Phase I Research Plan
The following Research Plan is proprietary and confidential information that Siskiyou Products requests not be released to persons outside the Government, except for purposes of review and evaluation.
TECHNICAL DISCUSSION OF PROPOSED CONCEPT
3.0 Articular cartilage from patients with OA is characterized by decreased concentrations of proteoglycans and glycosaminoglycans, as well as by a decreased size of glycosaminoglycan molecules1. In addition to these physical changes, osteoarthritic cartilage also undergoes certain quantitative changes involving concentration of species. Among these changes are a disproportionately increased ratio of chondroitin 4-sulfate to chondroitin 6-sulfate14, a decreased ratio of keratin sulfate to chondroitin sulfate14, and decreased sulfate ion of the terminal residues in chondroitin and dermatan sulfate chains15. Glycosaminoglycan molecules are well known and important sources of lubrication within the synovial capsule, affecting the viscosity of the synovial fluid itself. An acute depletion of HA and CS induced by intra-articular injection of HAase results in a drastic increase in synovial permeability of these molecules16. The reader is reminded that although we are concentrating on HAase in this study, it is also clearly recognized that degradation of the cartilage matrix that characterizes OA is a multifactorial process involving a number of enzymes, of which HAase is but one2. This Project will of course consider these and other factors in great detail, but will concentrate on shielding CS from the various enzymes that are active during times of osteoarthritic activity2. Specific testing will consist of in vitro analysis of how chondroiten sulfate (CS), hyaluronic acid (HA), and hyaluronidase (HAase) behave under a multitude of varied conditions involving pH, the molar concentrations of CS, HA, and HAase, and with the introduction of a number of experimental inhibitors9. Analytical methods will include Spectrophotometric, High Performance Liquid Chromatographic, and Gravimetric methods.
3.1 We anticipate experimenting with 25 to 200 different inhibitors. Generally, the successful Inhibitor to be utilized in this Project will have the properties and attributes described in §3.7 below. For purposes of this discussion, these inhibiting molecules will be collectively referred to as “Inhibitor.”
3.2 There exist 4 Stages of Experimentation to the successful conclusion of this Project to the point of bringing a Commercial Prototype to commercialization.
3.2.1 Stage 1 of this Project will determine those optimum Inhibitors that separately allow HA to be hydrolyzed in the presence of HAase and those Inhibitors that do not allow CS to be hydrolyzed when in the presence of HAase. Those Inhibitors from §3.1 will be initially identified by numerous methods and will have the characteristics defined in §3.7. We may utilize a newly developed software program described in §1.4, perform academic research into previous studies8, consider those that are known to interact with the negative polar site of the CS molecule (i.e., the double bonds of the SO4 constituent), consider the reactivity (however slight) of the CS and HA molecules with relatively inert inhibitors, and possibly engage in the synthesis of a new inhibitor altogether9. The Primary objective of the Phase I effort is to successfully identify these Inhibitors as stated in §2.1.A-D, which, after Stage 4, will become the Commercial Prototype to bring to the market. Without this identification, Stages 2-4 are impossible to accomplish. Stage 1 will be performed as part of Phase I.
3.2.2 Stage 2 of this Project will test and document each of the common Inhibitors arrived at in Stage 1, and then test their effect on the stability of HA, CS, and HAase (present together along with the Inhibitor) at varying pH levels and at varying concentrations of HA and CS. Depending upon the time required to complete Stage 1, Stage 2 may or may not be performed as part of Phase I.
3.2.3 Stage 3 will consist of in vitro testing of all of the Inhibitors found in Stage 2 in a simulated environment of the synovial capsule and its contents. In addition, Stage 3 will determine uptake of the Inhibitors into the various tissues with which the Inhibitors will have contact during their presence within the body. Stage 3 will further reduce the number of qualifying Inhibitors to those that meet all requirements as described in §3.7. Stage 3 will also involve testing the integrity of the CS-Inhibitor Intermediate against not only HAase, but also against hexosaminidase, sulfatases, glycosaminoglycanase, glycosidase and any enzymes usually found in OA synovial fluid. The mechanism5 by which the intermediate is formed between the Inhibitor and CS will be determined and clearly defined in this Stage while a commercial prototype of the Inhibitor that meets the requirements in §3.7 will finally be made towards the end of this Stage. Finally, Stage 3 will test the reactivity of the Inhibitors against those chemicals and cells existing in the blood stream and in the synovial capsule to ensure inert behavior. Remember: we are looking for an Inhibitor that is inert except against CS, when within the environment of synovial fluid, and only during increased pH levels. Stage 3 is not expected to be performed as part of Phase I.
3.2.4 Stage 4 will consist of in vivo testing of all of the Inhibitors from Stage 3 in rats or rabbits whose cartilage has been made osteoarthritic by laboratory-induced standard scarification techniques3. It is in this Stage where other enzymes will be present that hydrolyze mucopolysaccharides and glycosaminoglycans. The structural stability of CS that the Inhibitor brings by way of the CS-Inhibitor Intermediate bond that is formed will be tested in this stage. If CS remains intact as it did in Stage 3, HA is not accumulated within the synovial fluid, and the inhibitor is discarded and inert when the pH reaches normal levels, then we will have identified our Commercial Prototype as this Inhibitor. We will then proceed to test in another group of rats or rabbits whether the introduction of this Commercial Prototype into OA joints will not only shield CS from degradation, but whether that shielding results in the success of our primary goal of halting or reversing the degeneration of articular cartilage. If it does, then we have a marketable Commercial Prototype to present to one or more pharmaceutical companies for commercialization as is discussed in §4.10. Stage 4 is not expected to be performed as part of Phase I.
3.3 Stage 1 of this Project will begin by separately testing each Inhibitor with varying concentrations of HA and HAase present at varying pH levels so that we may determine which Inhibitor(s) will maximize the effect of HAase on the hydrolysis of HA (Fig. 1 below) because the Inhibitor we are looking for should selectively hydrolyze HA as if the Inhibitor was not present. Our main focus is to eventually determine an Inhibitor that will selectively hydrolyze HA but not CS. The Inhibitor should have no effect on the concentration or activity of HAase. Stage 1 will continue to §3.4. (See §3.2.1 for more discussion on Stage 1.)
3.4 Each Inhibitor will be separately tested with varying concentrations of CS and HAase present at varying pH levels so that we may determine which Inhibitor(s) will best loosely be attached via the electronegativity existing near the double bonds of the SO4 constituent of CS. The Inhibitor should have no effect on the concentration or activity of HAase. We will be looking for the Inhibitor that minimizes the effect of HAase on the hydrolysis of CS because of the loose attachment of the Inhibitor to the double bonds of the SO4 constituent. Because our main focus is to eventually determine an Inhibitor that will selectively hydrolyze HA but not CS, we are concerned with concentrating on the differences between HA and CS (See Figs. 1 and 2 below). The SO4 constituent of CS is therefore being chosen because it is that constituent which is the main difference between HA (Fig. 1 below) and CS (Fig. 2 below), and because of the presence of its double bonds which are by definition pre-disposed to chemical activity. This concept is foundational to the success of this entire Project.
Figures 1 and 2, showing the differences between Hyaluronic Acid and Chondroiten Sulfate:
Figure 1 C14H21NO11 Figure 2 C13H21NO15S
3.5 Those Inhibitors that are common to meeting the optimum requirements in §3.3 and §3.4 above and whose molecular size is within the limits of synovial membrane porosity will be used in Stage 2 of this Small Business Innovation Research Phase I Project, described in §3.6.
3.6 Stage 2 of this Project will test and document each of the common Inhibitors arrived at in §3.5, and then test their effect on HA, CS, and HAase (present together along with the Inhibitor) at varying pH levels and at varying concentrations of HA and CS. We are looking for the Inhibitor(s) that allow maximum hydrolysis of HA with HAase at high pH values and are effective in not allowing hydrolysis of CS with HAase at any pH value. Maximum hydrolysis of HA with HAase is defined as the hydrolysis of HA with HAase without the Inhibitor present. The successful Inhibitor should have no effect on the concentration or activity of HAase with all four species present, i.e., CS, HA, HAase, and the Inhibitor. (See §3.2 for more discussion on Stage 2.)
3.7 Selection of Inhibitors: Although the precise nature of the inhibitors we will select will be determined during the course of this Project as new parameters are uncovered and current parameters prove unnecessary, essentially, inhibitors will be organic molecules with limited polarity and will be chosen based upon their molecular size, positive polarity values, relative inert chemical reactivity, and immunity to absorption into various tissues. In addition, selection of the Inhibitor must take into consideration the viscosity and general composition of the synovial fluid in order to ensure eventual contact with patellar cartilage, and the porosity of the synovial capsule’s membrane if the method of delivery is through the blood stream rather than via intrusive injections.
The fundamental premise of this Project defines an Intermediate species formed by a partial bond between the SO4 group of CS and the Inhibitor. This concept has already been demonstrated among enzymes for it has been postulated that hexosaminidase hydrolyzes the β-glycosidic linkage of β-N-acetylglucosamine or β-N-acetylgalactosamine17. According to B.G. Winchester, an oxonium ion transition state is thought to be developed during the process. The partial positive polarity on the ring oxygen atom is stabilized by the deprotonated carboxyl group from the enzyme. This “partial bonding” behavior, already demonstrated to occur within osteoarthritic joints, gives credibility to the fundamental premise of this Project, where we will strive to form an intermediate with our inhibitor and CS across the double bond of the sulfate group. To test the strength of this intermediate, we may introduce glycoside sulfatases to our in vitro system4, and monitor how well the sulfate group withstands its attack.
In addition to forming a partial bond across the SO4 constituent, the Intermediate formed by the Inhibitor and CS as a result of this bond must be such (by size or chemical composition) that the action of HAase on the specific hydroxyl groups of CS that normally result in hydrolysis is blocked.
One method of determining the nature and identity of an inhibitor consists of utilizing previous studies such as those on the glycosidases and glycoside sulfatases secreted naturally by human articular chondrocytes in the midst of osteoarthritic inflammation4. The main difference between what was found in these studies and what is required in this Project is that the bonding species with the Inhibitor should be the substrate rather than the enzyme. Another method of determining the inhibitor required may be the development of a software application to sift through the many known organic molecules in order to arrive at those that have the required attributes. Alternatively, a synthesis of a new molecule altogether to serve as our inhibitor may be in order9.
3.8 Quantitative Analysis: Spectrophotometric, High Performance Liquid Chromatographic, and Gravimetric methods of analysis will be performed in order to arrive at results for §3.3, §3.4, and §3.6. In each of those descriptions, we will test each Inhibitor at pH values from 7.2 - 8.0 in increments of .05 pH units against varying concentrations of each component involved in the test. The specific concentrations to be used will be determined based upon values existing in normal synovial fluid and values existing in osteoarthritic synovial fluid. In the analysis of cartilage tissue, histological methods will be used to qualitatively determine the extent of matrix degradation.
3.9 Control of pH: pH will be decreased by introducing HA until the desired pH is reached. In all cases, pH will begin at the maximum (a pH of 8.0) and slowly decrease in increments of .05 pH units until the desired pH is reached. At each increment, a sample will be taken for analysis according to §3.8.
3.10 Stage 3 will require an in vitro control group of a closed system emulating normal synovial fluid with normal patellar cartilage present. The in vitro test group will emulate osteoarthritic synovial fluid and cartilage whose CS constituent within its matrix has been degraded. The test group will contain high levels of HA and HAase. Both groups will be treated with the Inhibitors remaining from Stage 2, and analyzed according to §3.8 and §3.9. Results from this stage will further reduce the number of Inhibitors that qualify as described in §3.7. Only those Inhibitors that successfully inhibit the further degradation of articular cartilage in the presence of HA and HAase, and are of a molecular size that will allow permeability through the synovial membrane will be available for Stage 4 testing.
3.11 Those Inhibitors that successfully pass testing described in §3.10 above will be tested for their inert behavior against normal cells and chemicals present in blood and synovial fluid. In addition, their uptake into surrounding tissue will be monitored. As was performed during Stages 1 and 2, the effects upon existing chemicals, cells, and tissue will be analyzed via spectrophotometric, HPLC, and gravimetric methods. This investigation will most likely decrease the number of qualifying Inhibitors that meet the requirements in §3.7.
3.12 Stage 4 will repeat Stage 3 test methods described in §3.10 and §3.11 in vivo with either rats or rabbits that are artificially induced with osteoarthritic conditions via standard scarification techniques3. Qualitative analysis via histological methods will determine the extent of cartilage degradation, while the quantitative methods described in §3.8 will used to obtain more precise results. Those Inhibitors that successfully pass this test will be the final product of this Project and defined as our Commercial Prototype. However, it should be noted that the Commercial Prototype of this Project, even though it will have been shown to halt or reverse osteoarthritic cartilage degeneration, will not successfully perform as such in all cases. Those subjects whose articular cartilage has degenerated to a point of no return will not be viable subjects for this procedure. The individuals in whose synovial capsules the Inhibitor may be introduced where little or no cartilage remain will not benefit from the intended effect of halting or reversing OA. The expected threshold whereby treatment with the final product from this Project will have little to no effect will be reported as part of Stage 4.
ACHIEVEMENT OF OBJECTIVES
3.13 As is discussed in §3.3, §3.6, §3.10, and §3.12 above, we will have reached our final objective for all Stages of this Project when all of the following have occurred:
A. (Phase I) We have found the Inhibitor(s) that allow maximum hydrolysis of HA by HAase at high pH values.
B. (Phase I) We have found the Inhibitor(s) to be effective in not allowing hydrolysis of CS by HAase at any pH value.
C. (Phase I) We have found the concentration of Inhibitor(s) not affected by the presence or activity of HAase.
D. (Phase I) We have found the Inhibitor(s) to have no effect on the concentration or activity of HAase with all four species present, i.e., CS, HA, HAase, and the Inhibitor. In addition, we have found A, B, and C above to be true with all four species present.
E. We have identified the mechanism by which the CS-Inhibitor intermediate is formed.
F. We have found the Inhibitor(s) to be of a molecular size that is within the limits of synovial membrane porosity.
G. We have found the Inhibitor(s) to be inert in all appropriate physiological environments and non-reactive with normal cells and normal chemicals present in blood, urine, and synovial fluid.
H. We have found the Inhibitors(s) not susceptible to uptake by normal tissues present with which they may come into contact. These tissues would include veins and arteries, the synovial capsule, fatty tissue, the bladder, kidney, and other tissues that the Inhibitor might encounter.
I. We have determined the delivery system by which the Inhibitor will be introduced into the synovial capsule.
J. We have determined the extent of cartilage degradation that can exist before treatment with the Inhibitor will have little or no effect.
3.14 Objectives for Phase I, described in §3.3 and §3.6 include those listed in §3.13 as A, B, C, and D. Objectives for §3.10 and §3.12 include those listed in §3.13 as A through J.
Part 4: Commercial Potential
4.0 OA is the most common joint disorder affecting millions of Americans, aged 25 and older. It has been estimated that over 12% of this group of Americans, about 21 million (some sources have this estimate as high as 30 million), have clinical signs and symptoms of OA11. The marketable product that will result from the successful completion of the R&D associated with this Project will be a prescribed course of treatment with our Commercial Prototype that will halt or reverse cartilage degeneration caused from normal wear and tear or from a major trauma at very low cost in comparison to surgically intrusive methods of treatment. Those millions of people who suffer from CP and OA to varying degrees will be the target population to which this product will be marketed nationwide. However, the introduction of a new medication to the U.S. commercial market is a long and complex process that may extend many years from the successful completion of this Project. We will need to partner with an existing pharmaceutical company and work with the U.S. Food and Drug Administration to bring the Commercial Prototype to actual market launch. The revenue required for these last stages of preparation for market launch may be provided by any pharmaceutical company with which we partner as well as from investors once the Commercial Prototype is identified and isolated as what will be referred to hitherto as our Product.
THE COMPANY AND TEAM
4.1 Siskiyou Products was formed in 1989 as a computer software business, and was replaced by its sister company, Cabinet Pro LLC, in 2011. Prior to 2011 Siskiyou Products brought in revenue equaling approximately $220,000 per year. Since 2011, Siskiyou Products has been without revenue and in the process of being re-designed and re-equipped from a computer software company to a company whose singular goal is to bring a biochemical solution to degenerative joint disease.
4.2 Siskiyou Products currently has two employees associated with it: Frank D. Jimenez, whose position is that of chemist, and Eliana Jimenez whose position is that of Administrator and Marketing Director. As is discussed in the Biographical Sketch, Frank D. Jimenez has been involved in University-sponsored research on the subject of degenerative joint disease, as well as in the development of computer software that will enable him to not only continue in the biochemical research he began at CSU Long Beach for this Project, but also to develop a software application to aid in the undertaking of Stage 1 of this Project (see §3.2.1). Eliana Jimenez has served as Administrator and Marketing Director of Siskiyou Products and Cabinet Pro LLC for 12 years, and will bring her organizational and marketing skills to the current vision of Siskiyou Products.
Eliana Jimenez has extensive experience in bringing a product to the marketplace. She has more than doubled the revenue generated by Siskiyou Products from $90,000 annual profit prior to her employment to $220K - $260K annual profit after her employment in 2001. Our business experience of over 25 years in successfully developing and marketing software products has demonstrated the need for distributorships, competitive pricing, and a viable marketing campaign, without which a product remains unknown.
We have been successful in developing, marketing, and maintaining our software products, and believe the business experience gained during that period has well prepared us for the new business endeavor defined by this proposed Project.
4.3 This Project will have an impact of creating a total of 6-7 new jobs, as well as utilize the services of outside consultants. Siskiyou Products plans to hire one additional employee for the completion of Phase I, and additional employees plus pharmaceutical consultants after Phase I. The responsibilities of these employees will include secretarial, custodial, data entry, academic research, animal care, and laboratory technician responsibilities. The pharmaceutical consultant(s) we plan to utilize will help in giving direction with an effective method of approaching existing pharmaceutical companies for the purpose of taking our product to the commercial marketplace. In addition, we plan to hire two additional employees to take over the responsibilities of our second company, Cabinet Pro LLC. These two employees, whose salaries will be paid by Cabinet Pro LLC’s revenue, will oversee technical support, programming enhancements, website maintenance, and general marketing of the existing software products available from Cabinet Pro LLC, thus allowing Frank D. Jimenez and Eliana Jimenez to devote themselves full time to Siskiyou Products and this Project.
PRODUCT AND COMPETITION:
4.4 The product we are seeking with this Project is a substance that, when introduced into the synovial capsule, will bond with CS under osteoarthritic conditions and thereby halt the degeneration of the main constituent responsible for cartilage integrity. Our goal is to halt or reverse OA, not to simply alleviate symptomatic pain. The product we are seeking with this Project will dissociate from CS and be inert under normal joint biochemical conditions. To this end is Siskiyou Products dedicated, for this is the sole mission statement of the company.
4.5 Even though the cost of OA has risen over recent decades12 and despite its widespread public health impact, the conservative treatment of OA is still limited to a few medications, such as salicylates, non-steroidal anti-inflammatory drugs, and corticosteroids that are injected directly into the joint. Hyaluronic Acid has also been used to alleviate symptomatic OA, but its positive effect is generally attributed to increased lubrication of the joint by synovial fluid, not from cartilage regeneration7. All of these medications provide primarily symptomatic pain relief and have not been demonstrated to halt or reverse the progression of the disease13. So the competition for a product that halts or reverses cartilage degradation that is found in degenerative joint disease is essentially non-existent. Ironically, it is the lack of competition that makes this entire endeavor a high risk undertaking (see §1.5).
4.6 One of the first tasks of Siskiyou Products prior to marketing the Inhibitor that is found as a result of the successful completion of this Project will be to patent not only the chemical structure of the Inhibitor, but also the mechanism by which the Inhibitor performs its task of halting or reversing degenerative joint disease. This requirement to Patent the mechanism we find to bond the inhibitor to CS in the formation of the intermediate species is the reason behind one of our major objectives sought at §2.1.E and found at §3.13.E.
4.7 Our vision is to bring our Commercial Prototype to the critical milestone of approaching one or more existing pharmaceutical companies for purposes of marketing that Prototype to the general public. The Product that will result from the successful completion of this Project will have similar commercialization precepts as those that have made our sister company successful, with the main difference being that these precepts will be concerned with marketing our Commercial Prototype to one or more pharmaceutical companies, rather than directly to the general public.
4.8 The Food and Drug Administration: Another critical milestone to meet well before the Commercial Prototype is brought to the marketplace is approval from the Food and Drug Administration. The FDA provides abundant online information on the task of bringing new drugs through the FDA process for final commercialization18. While it is not within the scope of this discussion to repeat that publicly available information here, some of the highlights should be mentioned.
4.8.1 The FDA requires an Investigational New Drug (IND) application to provide the data showing that it is reasonable to begin tests of a new drug on humans. Also, current Federal law requires that a drug be the subject of an approved marketing application before it is transported or distributed across state lines. Because we will want to ship the investigational drug to clinical investigators in many states, we must seek an exemption from that legal requirement. The IND is the means through which we will technically obtain this exemption from the FDA. During the early preclinical development for our Inhibitor, our primary goal is to determine if the product is reasonably safe for initial use in humans, and if the compound exhibits pharmacological activity that justifies commercial development. When our product is identified as a viable candidate for further development, we will then focus on collecting the data and information necessary to establish that the product will not expose humans to unreasonable risks when used in limited, early-stage clinical studies.
4.8.2 This section consists of direct quotes from the FDA concerning the various phases required for a new and untested drug. According to the FDA, the clinical investigation of a previously untested drug is generally divided into three phases. Although in general the phases are conducted sequentially, they may overlap. The three phases of an investigation are as follows:
Phase 1 includes the initial introduction of an investigational new drug into humans. These studies are usually conducted in healthy volunteer subjects. These studies are designed to determine the metabolic and pharmacological actions of the drug in humans, the side effects associated with increasing doses, and, if possible, to gain early evidence on effectiveness. Phase 1 studies also evaluate drug metabolism, structure-activity relationships, and the mechanism of action in humans. The total number of subjects included in Phase 1 studies is generally in the range of twenty to eighty.
Phase 2 includes the early controlled clinical studies conducted to obtain some preliminary data on the effectiveness of the drug for a particular indication or indications in patients with the disease or condition. This phase of testing also helps determine the common short-term side effects and risks associated with the drug. Phase 2 studies usually involve several hundred people.
Phase 3 studies are intended to gather the additional information about effectiveness and safety that is needed to evaluate the overall benefit-risk relationship of the drug. Phase 3 studies also provide an adequate basis for extrapolating the results to the general population and transmitting that information in the physician labeling. Phase 3 studies usually include several hundred to several thousand people.
These phases are just the beginning of a long and involved process required to bring a new drug to market with the approval of the FDA. With all due respect to the reader, we will not re-print the entire process here for two reasons. The first is that the entire process is readily available online18. The second reason is that we fully expect to relinquish this task to an already existing pharmaceutical company whose experience and expertise far outweighs our own in this area of working with FDA regulations.
4.9 Degenerative Joint Disease is the most common and wide spread joint disorder, affecting millions of Americans. To eradicate this disease in a large segment of this affected population would have a tremendously positive social impact on our society. For success in this current Project is not intended to merely alleviate the pain and symptoms associated with various forms of degenerative joint disease, but its scope is to halt or reverse the process of cartilage degeneration due to trauma or normal wear and tear that generally results in lesions of CP or OA. In doing so, the burden of treating those with degenerative joint disease for the medical community in general will be greatly relieved because the reversal of the lesion itself is far more effective than simply blocking pain. And the accomplishment of that reduction in burden to the medical community promises high commercial returns. Siskiyou Products will, at the end of this Project, bring the final product to commercialization by sub-contracting or selling to one or more pharmaceutical companies the rights to further test on human subjects, manufacture, market, and distribute the finished Commercial Prototype to the general public for profit.
FINANCING AND REVENUE MODEL
4.10 Commercial and Market Prospects: The path that represents by far the quickest and most direct plan for commercialization is for the Marketing Director of Siskiyou Products to approach already existing pharmaceutical companies as the obvious choice for obtaining FDA approval, manufacturing, packaging, marketing, and distributing the Commercial Prototype resulting from this Project.
The pharmaceutical companies we plan to approach for bringing our product to the market include, but are not limited to the following:
1. Johnson & Johnson
8. Abbott Laboratories
9. Merck & Co.
10. Bayer HealthCare
11. Eli Lilly
12. Bristol-Myers Squibb
Part 5: Consultants and Subawards/Subcontracts
5.0 Siskiyou Products will consult with the local university for purposes of academics, but will not sub-contract any work outside of Siskiyou Products for Phase I.
Part 6: Equivalent or Overlapping Proposals to Other Federal Agencies
Part 7: Lineage of the Innovation
telephone: (541) 664-2808