Inspire Pharmaceuticals, Inc.

Extracellular Nucleotides
Evidence for biological activity of extracellular nucleotides was documented as early as 1929, but it wasn't until 1970 when Professor Geoff Burnstock (member Inspire's Scientific Advisory Board) first put forth the notion that ATP was a non-adrenergic, non-cholinergic neurotransmitter. This radical idea, that ATP was not just an energy source for cells, was met with great resistance and skepticism, and many scientific debates were staged before this new field of biology matured with the cloning of the first P2Y nucleotide receptor in 1993. Now, an unimagined complex system involving a wide variety of extracellular nucleotides, their receptors, metabolizing enzymes and transporters, constitute one of the most exciting areas in cell signaling that is gaining attention on par with that of the adrenergic and cholinergic systems.

It is clear now that extracellular nucleotides regulate a wide variety of functional responses in many cell types by stimulation of both G-protein coupled receptors (P2Y) and ATP-gated ion channel receptors (P2X). To date, at least eight P2Y receptors and seven P2X receptors have been cloned from human tissue and their functional activities clearly demonstrated. Some members of the P2Y receptor family are selective for adenine nucleotides such as the P2Y1, P2Y11, P2Y12, and P2Y13, whereas other are selective for uridine nucleotides such as the P2Y4, P2Y6, and the UDP-glucose receptor. The P2Y2 receptor is activated by both adenine and uridine nucleotides. Among both purine- and pyrimidine-preferring receptors there are members with selectivity for nucleoside diphosphates over nucleoside triphosphates and vice-versa.

NUCLEOTIDE - A molecule of structure similar to building blocks of nucleic acids (DNA and RNA). Nucleotides via interaction with specific receptors stimulate various processes within the cell.

P2Y RECEPTOR - P2Y receptors are protein molecules expressed on the surface of cells. Receptors bind specific types of molecules called ligands (large number of marketed drugs are receptor ligands). This binding activates specific processes within the cell. A simple way to think of a drug binding to a receptor is the analogy of a key fitting a lock. A drug (a key) binds to a receptor (a lock) and activates specific processes within the cell. Inspire's drug candidates bind to a particular type of receptor called the P2Y2 receptor (one type of P2Y receptor) and activate the processes of mucosal hydration and mucociliary clearance.

HIGH THROUGHPUT SCREENING - A method for testing a large number of molecules for biological activity in very rapid fashion.

AGONIST - A chemical substance that activates ("turns on") certain activities in a cell by binding to and activating a receptor.

ANTAGONIST - The opposite of an agonist is an antagonist, which is a chemical substance that blocks or reduces ("turns off") certain activities in a cell by blocking the receptor.

Inspire's Screening Assay
Our primary screening platform is a cell-based assay using human P2Y and P2X receptors expressed into human astrocytoma cells. By coupling these receptors to intracellular calcium release, Inspire scientists were able to screen a number a chemical libraries for receptor agonists and antagonists. This assay is also used for selectivity, lead optimization and molecular pharmacology studies.

Inspire scientists, working with UNC researchers, immersed themselves in this emerging field, and were soon recognized as leaders in the discovery and synthesis of nucleotides that activate the P2Y2 receptor. Shortly after the initial technology was licensed
Inspire developed the first high throughput screen for P2Y receptor, capable of identifying both receptor agonists and antagonists. Inspire currently uses this assay for screening of small compound libraries, and has transferred the assay to contract laboratories for conducting high throughput screening with larger chemical libraries (i.e., > 100,000 compounds), and for screening that is not related to Inspire's core chemistry capabilities.

Nucleotide Therapeutics
Both purine and pyrimidine based nucleotides have been known to participate in a myriad of cellular functions for many years. One aspect of the biological utility of nucleotides arises from their actions as either agonists or antagonists of ion channel (P2X) or G protein coupled (P2Y) receptors. Even before the utility of nucleotides was discovered, science was aware of the therapeutic utility of purine analogues in the field of chemotherapy. George H. Hitchings and Gertrude B. Elion conducted some of the earliest work in the field of purine antimetabolites, which led to their being awarded the 1988 Nobel Prize in Medicine. 6-Mercaptopurine was one of the purine analogue antagonists that came out of their research and is still used today in chemotherapeutic applications. It was also hypothesized that pyrimidine bases could have utility as well as a chemotherapeutic, resulting in the discovery of 5-fluorouracil.

This early ground-breaking work set the stage for nucleoside and nucleotide therapeutics. Armed with the knowledge that nucleotides function as precursors of the nucleic acids, serve as energy stores, effectors, and group transfer agents, and mediate hormonal action, Inspire Pharmaceuticals is exploring and developing novel therapeutic dinucleotides for treatment of human diseases. Dinucleotides, such as diuridine tetraphosphate and diadenosine tetraphosphate (Up4U and Ap4A), show an improvement in chemical and metabolic stability over endogenous nucleotides while retaining activity at various P2 receptors, their primary targets. P2X or P2Y receptors are found in virtually every organ and tissue throughout the body. Epithelial tissue, in particular, expresses multiple subtypes of the P2Y family. Epithelial tissue comprises a layer or layers of cells that cover free and enclosed surfaces throughout the body. All epithelial layers contain two specialized domains: an apical domain that faces the mucosal (or external) space and a basolateral membrane that faces the serosal (or internal) space. Thus an important function of all epithelia is to provide an appropriate barrier function to separate and to control many physiological processes between these two spaces. Epithelial cells have evolved to serve many homeostatic functions that are specific to their location throughout the body. Cell surface receptors such as the P2Y family are involved with regulation of a portion of these functions. In the lung the airway epithelia serve many functions which include providing a barrier between the lung mucosa and blood supply, coordinating the hydration of the airways, regulating blood-borne immune responses in the airway mucosa, and clearing the airways of toxins and pathogens.

Dinucleotides
Inspire chemists have developed versatile strategies for the synthesis and purification of dinucleotides and dinucleotide derivatives. Inspire has discovered that dinucleotides with a proportional number of phosphates maintain the specificity and potency of their cognate mononucleotides, while enjoying enhanced resistance to ectonucleotidases and other nucleotide metabolizing enzymes. For example, Inspire lead compound, INS37217 has an EC50 of 9.8 x 10^-8M at the P2Y2-receptor, while its cognate INS316 has an EC50 of 9.3 x 10^-8M, and INS37217 persists longer than INS316.

Nucleotoids
Inspire is leveraging years of experience with P2 receptors and nucleotide chemistry toward the development of novel NUCLEOTOIDS. Using experimental and computational methods that correlate chemical structure with biological activity, Research and Preclinical Development scientists are designing these nucleotide-like substances for systemic administration, for example as ANTITHROMBOTIC DRUGS. NUCLEOTOIDS retain the properties of their parent nucleotides to agonize or antagonize P2 receptors, but have replacement of certain functional groups, such as phosphates, with alternative groups that impart improved overall solubility, stability, and oral bioavailability.

New Discoveries in Nucleotide Release, Metabolism & Re-Uptake
The landscape of nucleotide signalling has become increasingly clear and shows many similarities to other neurotransmitter signalling systems. In fact, purinergic signalling has been found to be a major component of most neurosensory units, and it is often referred to as the non-adrenergic, non-cholinergic, or "NANC," pathway. With this in mind, biological systems for the handling of extracellular nucleotides have evolved, including mechanisms for release, metabolism and re-uptake.

Although no proteins have been directly linked to nucleotide release, their catabolism by extracellular enzymes has been elucidated. Several families of ectonucleotidases are involved in the catabolism of released nucleotides, thus terminating or redirecting their activity at nucleotide receptors. In fact, knock out animals (genetically altered not to have a specific enzyme) of ectonucleotidases show interesting disease phenotypes that may be useful for targeting novel therapies.

The signal transduction of purinergic receptors is a growing field of research in which studies have shown potentiating effects of nucleotides on other receptor signalling responses, implying that purinergic receptors are involved in a host of downstream events that are not fully understood at present. Interestingly, the newly cloned P2Y11 receptor is a known G-protein coupled receptor that couples dually to Gs and Gq, resulting in divergent, parallel intracellular signals. The inter-relatedness of purinergic signalling to other systems serves a bridge into uncharted pathways of future disease targets and novel therapeutic approaches.