Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
  • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/16—Amides, e.g. hydroxamic acids
  • A61K31/18—Sulfonamides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
  • A61K31/255—Esters, e.g. nitroglycerine, selenocyanates of sulfoxy acids or sulfur analogues thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
  • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
  • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
  • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/415—1,2-Diazoles
  • A61K31/4155—1,2-Diazoles non condensed and containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents

Definitions

• the present invention relates to the use of one or more non-peptidyl compounds for the treatment of a patient suffering from one or more insulin related ailments.
• derived from shall be taken to indicate that a specific integer may be obtained from a particular source albeit not necessarily directly from that source.
• agents such as hormones and growth factors elicit their biological functions by binding to specific recognition sites, or receptors, in the plasma membranes of their target cells. This typically causes a conformational change in the receptor, and triggers secondary cellular responses which may result in the activation or inhibition of intracellular processes.
• agents are often referred to as biological modifiers. They may be categorised into two classes according to their activity. Agents which have a stimulatory activity are termed agonists, and those which inhibit the effect of the original ligand are termed antagonists.
• biological modifiers that differ in structure from the original ligand may be medically useful. Such compounds may have slightly different spectra of biological activity, allowing them to be used in very specific situations. Compounds that are chemically simpler than the agent which they mimic are potentially produced in greater quantities and at lower costs. Compounds that are more chemically robust than the original ligand may be administered by more convenient means. For example, peptidyl ligands cannot be taken orally as they will be broken down in the digestive tract, whereas a non-peptidyl agonist may potentially be administered by this route.
• Insulin is a peptidyl ligand, and the identification of agonists and antagonists thereof has important pharmaceutical applications in the treatment of insulin related ailments.
• Insulin is regarded as the most important regulatory hormone involved in maintaining glucose homeostasis. In vivo, insulin is produced in the pancreatic ⁇ - cells of the Islets of Langerhans. It is secreted from these cells in response to a wide range of nutrients, hormones and neurotransmitters. However, glucose is considered the most important regulator of insulin release. Following its release insulin is carried systemically to target tissues such as liver, muscle and fat. It promotes the uptake of glucose in the peripheral tissues and inhibits gluconeogenesis in the liver. Insulin also is involved in: promoting the synthesis of glycogen, lipid, and protein; gene transcription and mRNA turnover, and the transport of specific amino acids and ions.
• Insulin works by first binding to the insulin receptor on the cell surface of its target tissues.
• the insulin receptor is a specific transmembrane glycoprotein composed of two ⁇ -subunits and two ⁇ -subunits linked by disulfide bonds.
• the ⁇ -subunits are located extracellularly and contain the insulin binding domain.
• the ⁇ -subunits of the receptor pass through the plasma membrane and have an intrinsic tyrosine kinase activity associated with their intracellular domain.
• the X-ray coordinates of part of the tyrosine kinase domain has been resolved (1 , 2), however, the coordinates detailing the structure of the ⁇ -subunits are not yet available.
• Some inferences about the structure of some domains of the ⁇ -subunit can be made from fibronectin structures of homologous protein domains resolved by NMR spectroscopy and from the X-ray structure of the closely related IGF-1 receptor (3). Most recently, the general quaternary structure of the insulin receptor has been resolved using electron cryomicroscopy (4). These studies indicate that one insulin molecule probably binds and effectively crosslinks a L1 -cysteine rich domain of one ⁇ -subunit and the L2 domain of the other ⁇ -subunit. This viewpoint is supported by models derived from the complex kinetics of insulin binding and the models of receptor oligomerisation described below. Following insulin binding to the ⁇ -subunit, the ⁇ -subunit is autophosphorylated on specific tyrosine residues and this promotes the tyrosine kinase activity of the receptor.
• the simplest model proposes that the insulin receptor population is characterised by a single class of homogeneous, non-interacting binding sites and that insulin action is directly proportional to the fraction of these receptors that are occupied.
• the hormone (H) interacts with the receptor (R-) to result in the formation of a complex (HR-), with subsequent rapid dissociation (k.-). Stabilisation of the complex (HR.) is through conversion (k 2 ) to another state, from which hormone dissociation is slow (k_ 2 ).
• R. Corin and D. Donner (11) postulate that increasing occupancy time favours the formation of the second complex at the expense of the first.
• this model supports the kinetic data that identifies a constant association rate, but a dissociation rate which decreases with time. As a result of a decreased dissociation rate, the dissociation constant is also reduced, therefore providing an explanation for the presence of receptors of two apparent different affinities. It is not known if the bound complex HR 2 may dissociate directly to H and R 2 through k .3 and to R 1 through k_ 4 .
• the model proposes that insulin contains two binding surfaces on each ⁇ - subunit which can interact independently with corresponding surfaces on another ⁇ -subunit of the receptor. These binding sites, termed ⁇ 1 and ⁇ 2, have differing affinities with ⁇ 1 having a higher affinity than the ⁇ 2 region. If insulin binds to the ⁇ 1 site on one ⁇ -subunit, it is suggested that this induces a conformational change in the receptor which ultimately allows the insulin molecule to interact with the ⁇ 2 site on the second subunit. Consequently, the two ⁇ -subunits are cross- linked (homo dimerisation), resulting in high affinity binding between insulin and its receptor and subsequent activation of the receptor.
• Receptor oligorme ⁇ sation is a ubiquitous phenomenon among growth factor receptors It may be induced by monomeric ligands such as EGF to induce conformational changes that result in receptor-receptor interactions, or bivalent ligands (eg human growth hormone (hGH)) that mediate dime ⁇ sation of neighbouring receptors.
• monomeric ligands such as EGF to induce conformational changes that result in receptor-receptor interactions
• bivalent ligands eg human growth hormone (hGH)
• hGH human growth hormone
• the structure of subclass II receptors eg. insulin and IGF-1 are different because they exist as disulfide linked pairs of dimers in a heterotetrameric structure. In these cases, ligand binding to the receptor induces allosteric interaction between the two ⁇ halves to activate the receptors.
• insulin binding induces a conformational change in the insulin receptor that is responsible for receptor activation.
• a number of studies indicate that this conformational change may occur by a cross-linking mechanism, similar to that described above.
• insulin receptors are activated by a number of anti insulin receptor antibodies, but not by monovalent Fab' fragments, which implies a receptor cross-linking mechanism of activation similar to EGF receptors.
• the homobifunctional cross-linking agent disuccinimide suberate forms more ⁇ 2 species in the presence than in the absence of insulin, indicating that insulin draws the ⁇ -subunits into close proximity.
• reduced ⁇ dimer half receptors when immobilised on wheat germ agglutinin Sepharose do not phosphorylate in the presence of insulin. However, when they are free to associate in solution, insulin causes phosphorylation of the ⁇ -subunits, indicating that cross-linking of receptor ⁇ halves must occur for receptor activation.
• concanavalin A induces insulin receptor activation by cross-linking receptors in intact cells, but when monovalent, only slightly induces receptor activation.
• the dimeric insulin receptor can bind two (or more) insulin molecules (compared with growth hormone which cross-links two hGH receptors) the first insulin molecule binds more tightly than the second molecule and maybe all that is physiologically relevant for receptor activation.
• insulin receptor activation may well be synonymous with hGH activation of its receptor (or similar cytokine/hormone receptor activation). Indeed, the increased rate of dissociation of tracer insulin in the presence of unlabelied insulin has been explained by analogy to the self-antagonism seen for hGH and its receptors.
• Insulin binding to the two receptor ⁇ -subunits causes activation of the receptor ⁇ - subunit tyrosine kinase activity which leads to the phosphorylation of various substrates of insulin action.
• IRS insulin receptor substrates
• At present four members of the IRS family have been discovered. It is believed that these molecules have the potential to interact with, and thereby activate, other downstream signalling molecules, leading to many of the actions of insulin.
• the signalling pathways regulated by the individual substrates may vary.
• Diabetes is a complex disease with many causative factors. Hyperglycemia is a major characteristic of the disease and over time, especially if poorly controlled, leads to many complications of the disease. These complications include microvascular and macrovascular diseases, retinopathy, neuropathy, stroke, hypertension, heart and kidney disease. Careful control of blood glucose levels is, therefore, a key strategy in treatment of diabetes.
• IDDM insulin-dependent diabetes mellitus
• IDDM insulin-dependent diabetes mellitus
• the symptoms of IDDM include polyuria, polyphagia, polydipsia, weight loss and drowsiness.
• IDDM patients are absolutely dependent on regular injections of insulin for survival. Over 20 million people worldwide are dependent upon insulin in this manner.
• Several different types of human insulin are commercially available for diabetics, ranging from the fast-acting HumulinTMBR and NovolinTM to slower acting treatments, such as Protamine-zinc-insulin (PZI), Neutral protamine Hagedorn (NPH) insulin and Lente insulin. Insulin analogues like Humalog (LysPro) with altered properties are also available.
• PZI Protamine-zinc-insulin
• NPH Neutral protamine Hagedorn
• Lente insulin Insulin analogues like Humalog (LysPro) with altered properties are also available.
• Insulin analogues like Humalog (LysPro) with altered properties are also available.
• Each of these insulin therapies have their advantages and disadvantages and so the choice of insulin therapy should be made by the patient and physician with all information about the patient's lifestyle, physical performance, and drug preferences.
• NIDDM non-insulin dependent diabetes mellitus
• NIDDM insulin receptor substrate
• the sulfonylureas are the most common oral hypoglycemics and are traditionally used in the treatment of non-obese sufferers of NIDDM. They promote the beta- cells in the Islets of Langerhans of the pancreas to secrete insulin and so they effectively augment glucose-induced insulin secretion.
• the biguanide class of oral hypoglycemic agents increase insulin sensitivity and therefore can be used to lower blood glucose levels in NIDDM. Their mechanism of action is unclear. Phenformin is no longer used to treat NIDDM as it can cause fatal lactic acidosis. Metformin now is the only biguanide in clinical use worldwide. It is most commonly used when dietary therapy is unsuccessfully used to regulate blood glucose levels in obese patients. Metformin may be used in conjunction with sulfonylureas in instances where sulfonylurea therapy alone is inadequate or it may be used in combination with insulin in the treatment of IDDM. However, the adverse side-effects of metformin therapy may include lactic acidosis, nausea, bloating, diarrhoea and abdominal cramping.
• ⁇ -Glucosidase inhibitors are also used to treat NIDDM as an adjunct to dietary measures or sulfonylureas therapy. These compounds allow carbohydrate in the gut to be processed more effectively by slowing down their absorption from the intestinal tract. Adverse side effects of these compounds include flatulence, diarrhoea and abdominal pain.
• the thiazolidinediones are another class of compounds that may ameliorate symptoms of NIDDM. These compounds work by reducing insulin resistance at the sites of insulin action in the muscle and liver. They may be used in combination with insulin or sulfonylurea drugs but are not recommended for the treatment of IDDM. Severe adverse side-effects are rarely observed, however, some NIDDM patients fail to respond to this treatment.
• Insulin or hypoglycaemic drug overdose are clinical conditions that are often difficult to manage and may require hospitalisation and several days of intensive care. Non-accidental overdose or suicide attempts are fairly rare but often lead to death or profound neurologic impairment. The key symptoms of insulin overdose are hypoglycaemia, hypokalaemia and acid-base imbalance. Sulfonylurea drug overdose predominantly causes hypoglycaemia.
• Insulinomas account for about 90% of all pancreatic endocrine tumours. They occur with an incidence of about 0.5 per million population and people of all ages can be affected. Early diagnosis and treatment of insulinomas is essential because of their variable manifestations and potential lethality. These tumours are usually benign but synthesise and secrete insulin autonomously causing spontaneous hypoglycaemia. Symptoms may include deep coma, epilepsy, dizziness, weakness, hunger and epigastric pain.
• Congenital hyperinsulinism is the most common cause of severe, persistent hypoglycaemia in infants. It may be familial as up to 20% of affected families have more than one affected child. A defect in beta-cell function is the most likely explanation for the hyperinsulinism that can lead to brain damage and death if not detected early.
• Gastric dumping syndrome is encountered in approximately 25-50% of patients following gastric surgery and may persist post-operatively for several months. Early dumping usually involves gastro-intestinal and vasomotor complaints. Late dumping predominantly involves vasomotor complaints and is a consequence of a reactive hypoglycaemia resulting from hyperinsulinism and an exaggerated release of glucagon-like peptide-1. The gastric dumping syndrome is infrequently reported in children, but is difficult to diagnose and manage and has significant morbidity.
• hypoglycaemia secondary to the hyperinsulinism and characteristic of clinical conditions like those described above, be managed quickly if death or profound neurologic impairment is to be avoided.
• the mainstay of therapy in the management of severe hypoglycaemia is glucose or dextrose infusion. In cases of drug overdose, this may follow gut decontamination. Glucagon is used sometimes but considerable caution must be taken because its success depends on limited hepatic glycogen stores. Surgical intervention is quite successful for many insulinomas where the lesion can be appropriately localised. In the case of congenital hyperinsulinism, partial or complete pancreotectomy is often necessary. Surgical excision of injection sites in cases of massive, non-accidental insulin overdose also is sometimes the preferred option.
• Diazoxide is sometimes used to combat hyperinsulinism. It is a potent antihypertensive agent and acts to promote blood glucose level by suppressing insulin secretion from the pancreas. However, this drug is not specific in its treatment of hyperinsulinism and a number of undesirable side effects may follow its use. Hypotension, nausea, vomiting, dizziness, weakness and mild liver damage have been reported with its use. Severe hypoglycaemia also persists in some patients following diazoxide therapy.
• Octreotide is a peptide analog of somatostatin and, like diazoxide, one of its actions is to inhibit insulin secretion from the pancreas.
• Octreotide has shown some promise in treating patients with hyperinsulinism, however, resistance to the drug has been reported in some patients with insulinoma. It has been used with some success in treating gastric dumping syndrome in patients refractory to standard therapy. However, its long term use is limited by side-effects such as diarrhoea and steatorrhoea.
• octreotide may in some instances worsen existing hypoglycaemia by suppressing glucagon and growth hormone in the presence of unresponsive pancreatic hyperinsulinism.
• Octreotide therapy may also have undesired effects on reducing long term growth in infants.
• the only drugs available for treating hyperinsulinism with secondary hypoglycaemia act by suppressing further insulin secretion from the pancreas. No other drugs are presently available.
• Antagonists of insulin action would be therapeutically useful agents for treating a range of diseases or clinical conditions involving hyperinsulinism and hypoglycaemia. They would work by directly competing with insulin for binding to the insulin receptor (the first step of insulin action) and would thereby counter the effects of hyperinsulinism. Their effect would be to reduce the hypoglycaemic action of insulin evident in clinical conditions described above.
• the present invention relates to the use of at least a non-peptidyl compound as a biological modulator of insulin activity or insulin-related activity, which compound possesses ionic and hydrophobic chemical moieties spatially located so as to mimic at least an ionic and hydrophobic amino acid residue of insulin, which amino acids are associated with the binding of insulin to its receptor.
• Compounds of the present invention exert their effects by mimicing amino acids spatially located on insulin, enabling those compounds to bind to the insulin receptor or a like receptor causing biological modulation of the activity of the receptor.
• Compounds used in the present invention may act either as agonists or antagonists of insulin or insulin-like activity.
• the invention resides in a method for treating a patient suffering from one or more insulin related ailments, which method comprises the step of: administering to a patient an therapeutically effective amount of a compound that is a biological modulator of insulin activity, which compound possesses ionic and hydrophobic chemical moieties spatially located so as to mimic at least an ionic and hydrophobic amino acid residue of insulin, which amino acids are associated with the binding of insulin to its receptor.
• the present invention resides in the use of at least a non-peptidyl compound, which is at least a biological modulator of insulin activity and possess ionic and hydrophobic chemical moieties spatially located so as to mimic at least an ionic and hydrophobic amino acid residue of insulin, which amino acids are associated with the binding of insulin to its receptor; in the preparation of a medicament for the treatment of a patient suffering from one or more insulin related ailments.
• a method for identifying a non-peptidyl compound possessing ionic and hydrophobic chemical moieties spatially located so as to mimic particular ionic and hydrophobic amino acid residues of insulin which are associated with the binding of insulin to its receptor comprising the steps of: (1 ) comparing the three dimensional structure of the non- peptidyl compound with a three dimensional pharmacophore of an active site of insulin; and (2) selecting a non-peptidyl compound with ionic and hydrophobic chemical moieties spatially located so as to mimic said site.
• a method for determining whether a non-peptidyl compound identified by the third embodiment of the invention is an agonist or an antagonist comprising the step of: exposing the compound to an insulin or insulin like receptor and measuring the change in biological activity following exposure of the compound to the receptor.
• the present invention also contemplates novel chemical compounds identified by the method of the present invention as well as the use of those molecules in a pharmaceutical composition.
• the pharmaceutical composition comprising a chemical compounds capable of modulating the biological activity of insulin or and a pharmaceutically acceptable carrier and/or diluent.
• Figure 1 shows a receptor binding pharmacophore of insulin.
• Figure 2 tabulates a series of hypotheses generated from the insulin pharmacophore of Figure 1.
• Figure 3 tabulates features and tolerances of the hypotheses of Figure 2 used in the searching of three dimensional chemical databases.
• Figure 4 tabulates hypotheses and tolerances found to identify IM 175 in database searches, where the tolerance sphere for all features of the listed hypotheses was the size indicated in the body of the table.
• Figure 5 illustrates dose response of assays versus insulin concentration, showing typical dose response curves of Glucose transport (+), PTK assay (x) and Autophosphorylation experiments (o), which are expressed as a percent of maximal assay activation over a range of insulin concentrations.
• 125 plasma membranes (o) that is expressed as a fraction of total I insulin added, and where each data point is the mean of a triplicate determination.
• Figure 7 demonstrates the effect of IM 129 on I insulin bound to WGA.IR (o)
• Figure 8 illustrates the effect of IM 129 on total (in the presence of 50 nM insulin)
• Figure 9 shows the effect of IM 129 on total (presence of 2 nM insulin) (+)
• Figure 10 represents a proposed model of antagonist competition with insulin for binding to insulin receptors, in which two alpha-beta halves of the insulin receptor joined by disulfide bonds are illustrated, activated receptors are phosphorylated on their ⁇ -subunits insulin is illustrated by oblong shaped objects and in which antagonist molecules are illustrated by black circular objects
• Figure 11 shows the effect of IM 129 on I IGF-1 bound to human placental
• 125 plasma membranes which is expressed as a fraction of total I IGF-1 added and is overlayed with a 1-s ⁇ te fit to the data (solid line) and where each data point is the mean of a triplicate determination
• Figure 12 illustrates the effect of IM 025 on I insulin bound to human placental
• Figure 14 illustrates the effect of IM 025 on I insulin bound to WGA IR (o)
• Figure 15 demonstrates P incorporation into FYF peptide where the effect of IM
• Figure 16 represents the effect of IM 025 on total (presence of 2 nM insulin) (+)
• Figure 17 shows the effect of IM 071 on I insulin bound to human placental
• 125 plasma membranes (o) expressed as a fraction of total I insulin added where each data point is the mean of a triplicate determination.
• Figure 18 summarises the effect of IM 127 on I insulin bound to human
• 125 placental plasma membranes (+) expressed as a fraction of total I insulin added, where each data point is the mean of a triplicate determination.
• Figure 19 shows the effect of IM 127 on I insulin bound to WGA.IR (+)
• Figure 20 illustrates P incorporation into FYF peptide in the presence of IM 127, where the effect of IM 127 on total (in the presence of 50 nM insulin) (+) and basal
• Figure 21 demonstrates the effect of IM 127 on total (presence of 2 nM insulin)
• (+) and basal (no insulin) (o) H-deoxyglucose uptake by 3T3L1 cells, where glucose transport in is expressed as a percentage of a maximal 100 nM dose of insulin and where each point is the mean of a triplicate determination ⁇ one standard deviation.
• Figure 22 represents the effect of IM 132 on I insulin bound to human placental
• 125 plasma membranes (o), expressed as a percentage of total I insulin added, where each data point is the mean of a triplicate determination ⁇ one standard deviation.
• Figure 23 shows the effect of IM 132 on 1 5
• Figure 25 shows the effect of IM 132 on total (presence of 2 nM insulin) (+)
• Figure 26 shows the effect of IM 134 on I insulin bound to human placental
• 125 plasma membranes (o), expressed as a percentage of total I insulin added and where each point is the mean of a triplicate determination ⁇ one standard deviation.
• Figure 27 demonstrates the effect of IM 134 on total (in the presence of 400 nM
• Figure 28 shows the effect of IM 134 on H-deoxyglucose uptake in the presence (+) and absence (o) of 2 nM insulin by 3T3L1 adipocyte cells, where glucose transport in is expressed as a percentage of a maximal 100 nM dose of insulin, and each point is the mean of a triplicate determination ⁇ one standard deviation.
• Figure 29 illustrates the effect of IM 143 on I insulin bound to human placental
• 125 plasma membranes (o), expressed as a percentage of total I insulin added and where each point is the mean of a triplicate determination ⁇ one standard deviation.
• Figure 30 represents the effect of IM 143 on total (presence of 2 nM insulin) (+)
• Figure 31 shows the effect of IM 144 on I insulin bound to human placental
• 125 plasma membranes (o), expressed as a percentage of total I insulin added and where each point is the mean of a triplicate determination ⁇ one standard deviation.
• Figure 32 illustrates the effect of IM 144 on I insulin bound to WGA.IR (o),
• Figure 33 represents the effect of IM 144 on total (in the presence of 50 nM
• Figure 34 shows the effect of IM 144 on total (presence of 2 nM insulin) (+)
• 3 basal (no insulin) (o) H-deoxyglucose uptake by 3T3L1 cells, in which glucose transport in is expressed as a percentage of a maximal 100 nM dose of insulin, and where each point is the mean of a triplicate determination ⁇ one standard deviation.
• Figure 35 illustrates the effect of IM 145 on I insulin bound to human placental
• 125 plasma membranes (o), expressed as a percentage of total I insulin added and where each point is the mean of a triplicate determination.
• Figure 36 shows the effect of IM 145 on total (in the presence of 50 nM insulin)
• (+) and basal (no insulin) (o) H-deoxyglucose uptake by 3T3L1 cells, where glucose transport is expressed as a percentage of a maximal 100 nM dose of insulin, and each point is the mean of a triplicate determination ⁇ one standard deviation.
• Figure 38 represents the effect of IM 171 on I insulin bound to human placental
• 125 plasma membranes (o), expressed as a percentage of total I insulin added and where each point is the mean of a triplicate determination ⁇ one standard deviation.
• Figure 39 shows the effect of IM 171 on total (in the presence of 400 nM insulin)
• Figure 40 illustrates the effect of IM 171 on total (presence of 2 nM insulin) (+)
• Figure 41 shows the effect of IM 172 on I insulin bound to human placental
• 125 plasma membranes (o), expressed as a percentage of total I insulin added and where each point is the mean of a triplicate determination ⁇ one standard deviation.
• Figure 42 shows the effect of IM 172 on total (in the presence of 400 nM insulin)
• Figure 43 illustrates the effect of IM 172 on total (in the presence of 400 nM
• Figure 44 shows the effect of IM 172 on total (presence of 2 nM insulin) (+)
• 3 basal (no insulin) (o) H-deoxyglucose uptake by 3T3L1 cells, in which glucose transport in is expressed as a percentage of a maximal 100 nM dose of insulin, and where each point is the mean of a triplicate determination ⁇ one standard deviation.
• Figure 45 demonstrates the effect of IM 140 on I insulin bound to human
• 125 placental plasma membranes (o), expressed as a fraction of total I insulin added, where each data point is the mean of a triplicate determination.
• Figure 46 represents the effect of IM 140 on I insulin bound to WGA.IR (o),
• Figure 47 shows the effect of IM 140 on total (presence of 2 nM insulin) (+)
• Figure 48 shows the effect of IM 140 on total (presence of 2 nM insulin) (+)
• Figure 49 illustrates the effect of IM 140 on I insulin binding (+) and H- deoxyglucose (o) in 3T3L1 adipocyte cells, where each isotherm is expressed as a percentage of buffer control and each point is the mean of a triplicate determination ⁇ one standard deviation.
• Figure 50 represents a possible mechanism for agonist activation of insulin receptors, in which two alpha-beta halves of the insulin receptor, joined by disulfide bonds are illustrated, activated receptors are phosphorylated on their ⁇ - subunits, insulin is represented by oblong shaped objects and agonist molecules are represented by dumbbell shaped objects.
• Figure 51 demonstrates the effect of IM 140 on P incorporation into FYF peptide
• Figure 52 demonstrates the effect of IM 140 on the total (in the presence of 100
• Figure 53 illustrates the effect of IM 140 on blood glucose levels in streptozotocin induced diabetic mice.
• the scatter plot shows the effect of IM 140 at 21 ⁇ mol/kg (+), 30 ⁇ mol/kg (o), 37 ⁇ mol/kg (x) and human insulin at 2U/kg over a period of 40 minutes.
• a significant change in blood glucose levels in an unpaired 1-tail t-test are indicated by a single asterisk where p ⁇ 0.05, a double asterisk where p ⁇ 0.01 and a triple asterisk *** where p ⁇ 0.001.
• Figure 54 illustrates the effect of IM 140 on I IGF-1 bound (o) and I insulin bound (+) to human placental plasma membranes expressed as a fraction of total iinnssuulliinn aacdded respectively, where each data point is the mean of a triplicate determination.
• Figure 55 illustrates the effect of IM 140 on 125
• 125 plasma membranes (o), expressed as a fraction of total I insulin added and overlayed with a 1-site fit to the data (solid line).
• Figure 57 represents the effect of IM 175 on I IGF-1 bound to human placental
• 125 plasma membranes (o), expressed as a fraction of total I IGF-1 added and overlayed with a 1-site fit to the data (solid line), where each data point is the mean of a triplicate determination.
• Figure 58 is illustrative of the effect of IM 175 on total (presence of 2 nM insulin) (+) and basal (no insulin) (o) H-deoxyglucose uptake by 3T3L1 cells expressed
• Figure 59 demonstrates the incorporation of P into the 90 kDa protein band of immunoprecipitated insulin receptors, where the uppermost autoradiograph demonstrates phosphorylation of immunoprecipitated insulin receptor ⁇ -subunit in response to the indicated concentrations of IM 175 and insulin.
• Untreated lanes show phosphorylation in the absence of both insulin and IM 175, where both autoradiographs were taken from the same experiment, the leftmost (A) being a 48 hour exposure of the gel to X-ray film, and the rightmost (B) a 24 hour
• the lowermost plot demonstrates the effect of IM 175 on P incorporation into the 90 kDa protein band of immunoprecipitated insulin receptors in the presence (o) and absence (x) of 5 nM insulin compared to a 100 nM dose of insulin (+).
• Figure 60 demonstrates the effect of IM 175 on the incorporation of P into the 90 kDa protein band of immunoprecipitated IGF-1 receptors in the presence (+) and absence (x) of 2 nM IGF-1.
• Figure 61 shows the effect of IM 103 on I insulin bound to human placental
• 125 plasma membranes (o), expressed as a fraction of total I insulin added, where each data point is the mean of a triplicate determination.
• Figure 62 illustrates the effect of IM 103 on I insulin bound to CHO.T11 cells
• Figure 64 shows the effect of IM 103 on total (presence of 2 nM insulin) (+)
• Figure 65 illustrates The effect of IM 103 on I IGF-1 bound to human placental
• 125 plasma membranes expressed as a fraction of total I IGF-1 added and overlayed with a 1-site fit to the data (solid line), where each data point is the mean of a triplicate determination.
• the present invention relates to the use of at least a non-peptidyl compound as a biological modulator of insulin activity or insulin-related activity, which compound possesses ionic and hydrophobic chemical moieties spatially located so as to mimic at least an ionic and hydrophobic amino acid residue of insulin, which amino acids are associated with the binding of insulin to its receptor.
• the invention resides in a method for treating a patient suffering from one or more insulin related ailments, which method comprises the step of: administering to a patient an therapeutically effective amount of a compound that is a biological modulator of insulin activity, which compound possesses ionic and hydrophobic chemical moieties spatially located so as to mimic at least an ionic and hydrophobic amino acid residue of insulin, which amino acids are associated with the binding of insulin to its receptor.
• the present invention resides in the use of at least a non-peptidyl compound, which is at least a biological modulator of insulin activity and possess ionic and hydrophobic chemical moieties spatially located so as to mimic at least an ionic and hydrophobic amino acid residue of insulin, which amino acids are associated with the binding of insulin to its receptor; in the preparation of a medicament for the treatment of a patient suffering from one or more insulin related ailments
• biological modulator refers to a compound that is capable of least varying insulin activity when introduced into a biological system (eg an in vitro or in vivo system) Such a compound may either be an antagonist or an agonist of biological activity Most preferably the non-peptidyl compound used in either the first or second embodiment of the invention is an insulin agonist
• the term "patient” refers to any animal that may be suffering from one or more insulin related ailments Most preferably the animal is a mammal
• the term 'mammal refers to a class of vertebrates whose young feed upon milk from the mother's breast Most species are more or less hairy, all have a diaphragm, and all (except the monotremes) are viviparous
• the term will be understood to include for example human, farm animals (i e , cattle, horses, goats, sheep and pigs), household pets (i e , cats and dogs) and the like
• terapéuticaally effective amount refers to an amount of a non-peptidyl compound sufficient to modulate a biological activity associated with the interaction of insulin with its receptors
• Insulin related ailments include ailments which are related to decreased secretion of insulin decreased responsiveness of cells to insulin, or increased secretion of insulin and may, for example, include ailments such as - diabetes mellitus (types 1 and 2) insulinomas, insulin and hypoglycaemic drug overdose, gastric dumping syndrome and congenital hyperinsulinism Other ailments will be known to those of ordinary skill in the field For example, insulin and glucose therapy leads to improved cognition in alzheimers disease patients
• Ammo acids associated with the binding of insulin to its receptor include, by way of example - A21 Asn, B21 Glu, A17 Glu, B24 Phe, B25 Phe, A19 Tyr, B12 Val, B16 Tyr A2 lie, A3 Val and A1 Gly
• the "A” or "B” nomenclature refers to either the A or B chain that forms insulin, while the number identifies the ammo acid number in either chain
• the non-peptidyl compounds employed in the present invention may mimic any number of the amino acids associated with the binding of insulin to its receptor, preferably they mimic ionic and hydrophobic moieties associated with at least two of these amino acids.
• the compounds mimic ionic and hydrophobic residues on at least 4 of the abovementioned amino acids, including at least one amino acid from the group comprising:- A21 Asn, B21 Glu and A17 Glu. Even more preferably, the compounds mimic ionic and hydrophobic residues on at least 4 amino acids, including at least one amino acid selected from the group comprising A17 Glu, B21 Glu and A21 Asn, and at least one amino acid selected from the group comprising:- B24 Phe, B25 Phe, A19 Tyr, B12 Val and B12 Tyr. Desirably, the non-peptidyl compound mimics ionic and hydrophobic residues associated with at least one of the following groups of amino acid residues:-
• non- peptidyl compound(s) has the following formula:
• A is W or VXW
• V is V- or V 2 ;
• V is substituted with up to two X groups; V, is a phenyl or 6 membered heteroaromatic ring, optionally substituted with up to 5 R 1 groups for example but not being limited to benzene, pyridine, py ⁇ dazine, py ⁇ midine, pyrazine, t ⁇ azine,
• V 2 is a 5 member ring system which may incorporate up to 4 hetero atoms which may be independently a nitrogen atom, a nitrogen atom optionally substituted with R 2 , oxygen or sulfur, for example but not being limited to cyclopenta-1 ,3-d ⁇ ene, pyrrole, furan, thiophene, oxazole, isoxazole, pyrazole, imidazole, thiazole, isothiazole or tnazole, the ring system being optionally substituted with up to 4 R- groups,
• W is substituted with up to two X groups
• W 2 is a fused bicyc c ring system comprising rings of 5 or 6 atoms, which may incorporate up to 4 hetero atoms, which may be independently a nitrogen atom, a nitrogen atom optionally substituted with R 2 , oxygen or sulfur the system being optionally substituted with up to seven R- groups and examples include, but are not limited to naphthalene, quinohne, isoquinol e, phthalazine naphthy ⁇ dine, quinoxalme, quinazohne, cinnoline, pte ⁇ dine, indole, benzothiophene, benzofuran, benzimidazole, dazole, benzoxazole, benzisooxazole, benzthiazole, benzisothiazole, pu ⁇ ne, mdoline, isomdoline,
• W 3 is -N(R 2 )R' 2 ,
• R 1 is independently H, OH, alkyl, alkenyl, alkynyl, alkoxy, alkanol, hydroxyalkoxy, haloalkyl, haloalkoxy, halogen, SH, thioalkyl, cyano (-CN), N(R 2 )R' 2 , phenyl, phenyl optionally substituted with up to five alkyl groups of
• R 2 and R' 2 are independently H, alkyl of 1 to 6 carbon atoms, alkenyl of 3 to
• R 2 and R' 2 can also be joined to form cyclic structures including, but not limited to pyrro dine, piperidme, hexahydro-1 H-azep ⁇ ne, morpholme or piperazine,
• R 3 is independently H, OH, alkyl, alkenyl, alkynyl, alkoxy, alkanol, hydroxyalkoxy, -R 4 N(R 2 )R' 2 , mesyl, triflouromesyl, -NHS0 2 CH 3 or -
• R 4 is independently a bond, alkyl, alkenyl or alkynyl
• R 5 is independently alkyl, alkenyl, alkynyl, alkoxy, alkanol, hydroxyalkoxy, Y is either Y 1 , Y 2 or Y 3 ⁇ Y is substituted with at least two, but optionally up to four X linking groups,
• Y- is a fused bicyclic ring system comprising rings of 5 or 6 atoms which may incorporate up to 4 hetero atoms, which may be independently a nitrogen atom, a nitrogen atom optionally substituted with R 2 , oxygen or sulfur, the ring system optionally independently incorporating a sulfoxide (SO), sulfone (S0 2 ) or carbonyl (CO) group and optionally up to seven R- groups, for example but not limited to croman, isochroman, benzofuran, cromene, 1 ,2,3,4-tetrahydronaphthalene, 1 ,4-d ⁇ hydronaphthalene, indan, indene, benzopipendine indolme, isoindolme, quinohne, isoquinolme, phthalazine, naphthy ⁇ dine, quinoxal e, quinazolme cinno ne or ptendine, couma ⁇ n or 2,3-d ⁇
• Y 2 is a 6 6 6 or a 6 5 6 fused tricyclic system which may incorporate up to 4 hetero atoms which may be independently a nitrogen atom, a nitrogen atom optionally substituted with R 2 , oxygen or sulfur, the ring system optionally independently incorporating a sulfoxide (SO), sulfone (S0 2 ) or carbonyl (CO) group and the ring system being substituted with at least two, but optionally up to four X linking groups and optionally up to seven R- groups and thus examples include, but are not limited to 9H-xanthone, 9H-xanthene, phenoxathnn, phenoxath ⁇ n-10-ox ⁇ de, phenoxath ⁇ n-10-d ⁇ ox ⁇ de, acndine, phenazine, phenothiazine, phenoxazine, phenoth ⁇ az ⁇ ne-5-ox ⁇ de, phenoth ⁇ az ⁇ ne-5-d
• Z is independently -R 6 COOH, -R 6 S0 3 H, -R 6 N0 2 , -R 6 S0 2 H, -R 6 S0 2 NHR 2 , -
• R 6 is independently a bond, alkyl, alkenyl, alkynyl, alkoxy, -CO(CH 2 ) n -, where n is an integer between 0 and 4, alkanoic, alkenoic or alkynoic, with the exception that where W 1 is an optionally substituted phenyl then Y. cannot be an optionally substituted phenyl
• alkyl refers to an alkane derived radical of between 1 and 6 carbon atoms unless otherwise defined, including straight, branched or cyclic alkane derived radicals, monovalent or bivalent alkane derived radicals in that it may be joined to one or two groups via any allowed bond to its carbon atoms
• straight or branched alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, /-butyl, -butyl, pentyl or hexyl and the cycloalkyl groups are cyclopentyl or cyclohexyl
• alkenyl refers to a hydrocarbon derived radical containing from 2 to 6 carbon atoms, unless otherwise defined and at least one carbon to carbon double bond, and includes straight, branched or cyclic hydrocarbon derived radicals, monovalent or bivalent hydrocarbon derived radicals in that such alkane derived radicals may be joined to one or two groups via any allowed bond to its carbon atoms.
• the alkenyl groups are ethenyl, propenyl, butenyl, cyclopentenyl, or cyclobutenyl.
• alkynyl refers to a hydrocarbon derived radical containing from 2 to 6 carbon atoms and at least one carbon to carbon triple bond and includes straight chained or branched hydrocarbon derived radicals, monovalent or bivalent hydrocarbon derived radicals in that such hydrocarbon derived radicals may be joined to one or two groups via any allowed bond to its carbon atoms.
• the alkynyl groups are ethynyl, propynyl or butynyl.
• alkoxy refers to an alkyl group of indicated carbon atoms, attached to other groups through an oxygen linkage (monovalent) or; to other groups through an oxygen linkage and a bond to any of its allowed carbon atoms (bivalent)
• hydroxyalkoxy refers to an alkoxy group substituted at one or more carbon atoms, with one or more hydroxyl groups.
• haloalkoxy refers to an alkoxy group substituted at one or more carbon atoms, with one or more halogen atoms
• the halogen substituents are fluorine, chlorine, bromine or iodine.
• alkanol refers to an alkyl group substituted at one or more carbon atoms, with one or more hydroxyl groups
• haloalkyl refers to an alkyl group substituted at one or more carbon atoms, with one or more halogen atoms.
• the halogen substituents are fluorine, chlorine, bromine or iodine
• thioalkyl refers to an alkyl group of indicated carbon atoms, attached to other groups through a sulfur linkage.
• alkanoic refers to an alkyl group of indicated carbon atoms, substituted at one carbon atom with a carboxylic acid group (-COOH).
• alkenoic refers to an alkenyl group of indicated carbon atoms, substituted at one carbon atom with a carboxylic acid group (-COOH).
• alkynoic refers to an alkynyl group of indicated carbon atoms, substituted at one carbon atom with a carboxylic acid group (-COOH).
• non-peptidyl compound employed in the invention has the structure of formula 1 above and V is V- or V 2 ⁇ then:
• V- is selected from the group consisting of, benzene, pyridine, pyridazine, pyrimidine, pyrazine or triazine and is optionally substituted with up to 5 R- groups
• V 2 is selected from the group consisting of, cyclopenta-1 ,3-diene, pyrrole, furan, thiophene, oxazole, isoxazole, pyrazole, imidazole, thiazole, isothiazole or triazole and is optionally substituted with up to 4 R- groups; and W is W 2 then
• W 2 is selected from the group consisting of naphthalene, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, indole, benzothiophene, benzofuran, benzimidazole, indazole, benzoxazole, benzisooxazole, benzthiazole, benzisothiazole, purine, indoline or isoindoline and is optionally substituted with up to seven R 1 groups; and Y is either Y- or Y 2 then Y 1 is selected from the group consisting of croman, isochroman, benzofuran, cromene, 1 ,2,3,4-tetrahydronaphthalene, 1 ,4- dihydronaphthalene, indan, indene, benzopiperidine, indoline, isoindoline, quinoline, isoquinoline,
• Y 2 is selected from the group consisting of 9H-xanthone, 9H-xanthene, phenoxathnn phenoxath ⁇ n-10-ox ⁇ de, phenoxath ⁇ n-10-d ⁇ ox ⁇ de, ac ⁇ dine, phenazine, phenothiazine, phenoxazine, phenoth ⁇ az ⁇ ne-5-ox ⁇ de, phenoth ⁇ az ⁇ ne-5-d ⁇ ox ⁇ de, th ⁇ athrene-5-d ⁇ ox ⁇ de, th ⁇ athrene-5-ox ⁇ de, carbazole, d ⁇ benzo[b,d]furan or d ⁇ benzo[b,d]th ⁇ ophene and is optionally substituted with up to seven R- groups
• non-peptidyl compound employed in the invention has the structure of formula 1 above and A is W or VXW then
• V is phenyl or pyrazole, optionally substituted with up to 5 R. groups,
• W is phenyl optionally substituted with up to 5 R 1 groups
• W 2 is naphthalene or quinoline optionally substituted with up to seven R groups wherein R- is independently H, OH, methyl, ethyl, propyl, nitro, methoxy, ethoxy, 2-hydroxyethoxy, chloro, fluoro or acetyl, W 3 is -N(R 2 )R 2 wherein R 2 is propyl,
• Y 2 is 9H-xanthone optionally substituted with up to seven R- groups wherein R- is independently H, OH, methyl, ethyl, propyl, nitro, methoxy, ethoxy, 2-hydroxyethoxy, chloro, fluoro or acetyl,
• Y 3 is phenyl optionally substituted with up to 5 R. groups wherein R- is independently H, OH, methyl ethyl, propyl, nitro, methoxy, ethoxy, 2- hydroxyethoxy, chloro, fluoro or acetyl, and Z is independently -R 6 COOH, -R 6 S0 3 H or -N-trifluoromesylsulfonamidate wherein R 6 is independently a bond or propyl
• the non-peptidyl compound(s) are selected from the group:
• IM 025 (herein referred to as IM 025);
• IM 103 4. 7-[(4-acetyl-3-hydroxy-2-propylphenyl)methoxy]-4-oxo-8-propyl-4/-/-1- benzopyran-2-carboxylic acid.
• IM 127 (herein referred to as IM 127);
• IM 132 (herein referred to as IM 132);
• IM 134 (herein referred to as IM 134);
• IM 140 8. 8-propyl-7-(quinol-2'-ylmethoxy)-3,4-dihydro-2/-/-1 -benzopyran-2-carboxylic acid
• IM 143 10 7-(naphth-2'-ylmethoxy)-8-propyl-3 4-d ⁇ hydro-2/-/-1 -benzopyran-2- carboxylic acid
• IM 172 (herein referred to as IM 172), or
• non-peptidyl compounds described herein may also be prepared as dimers or heterodimers of compounds of the above mentioned formula AXYXZ n where such compounds are joined through a X linking group by way of their V or W groups
• the compounds of the present invention include chemical derivatives that may be converted to the above mentioned compounds in vivo, such derivatives including but not being limited to esters and amides
• the present invention also contemplates pharmaceutical composition
• pharmaceutical composition comprising at least a chemical compound capable of modulating the biological activity of insulin and a pharmaceutically acceptable carrier and/or diluent
• the compounds selected for use in the invention are desirably prepared in a purified form suitable for administration to a patient Purification of such compounds may be achieved by any means known in the art, such as distillation, chromatographic means ere
• compositions may be formulated into therapeutics as neutral or salt forms
• Pharmaceutically acceptable salts include, for example, the acid addition salts (formed with any free am o groups of the compounds) and which are formed with inorganic acid such as hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, maleic and the like
• Salts formed with free acidic groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or iron (III) hydroxides, and such organic bases as isopropylamme, t ⁇ methylamine, 2-ethylam ⁇ no ethanol, histidine, procame and the like
• the composition may further comprise excipients that are pharmaceutically acceptable and compatible with the active ingredient Examples of excipients which may be used in such a formulation include water, saline, ethanol, dextrose, glycerol or the like, or combinations thereof If desired, the composition may also contain minor amounts of auxiliary substances
• compositions may be administered by injection, or prepared for oral, pulmonary, nasal or for any other form of administration
• the pharmaceutically acceptable compos ⁇ t ⁇ on(s) are administered, for example, intravenously, subcutaneously, intramuscularly, intraorbitally, ophthalmically, mtraventricularly, mtracranially, mtracapsularly, intraspmally, tracisternally, intraperitoneally buccal, rectally, vaginally, intranasally or by aerosol administration
• the mode of administration must, however, be at least suitable for the form in which the composition has been prepared
• the mode of administration for the most effective response may need to be determined empirically and the means of administration described below are given as examples, and do not limit the method of delivery of the composition of the present invention in any way All the above formulations are commonly used in the pharmaceutical industry and are commonly known to suitably qualified practitioners
• compositions can be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable nontoxic excipients and carriers and administered by any parenteral techniques such as subcutaneous, intravenous and mtraperitoneal injections
• parenteral techniques such as subcutaneous, intravenous and mtraperitoneal injections
• formulations may optionally contain one or more adjuvants
• the pharmaceutical forms suitable for mjectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile mjectable solutions or dispersion
• the compounds of the invention may be encapsulated in liposomes and delivered in mjectable solutions to assist their transport across cell membrane
• such preparations may contain constituents of self- assembling pore structures to facilitate transport across the cellular membrane
• the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils
• Proper fluidity may be maintained, for example, by the use of a coating such as hcithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants
• Prolonged absorption of the mjectable compositions can be brought about by the use in the compositions of agents
• Sterile mjectable solutions may be prepared by incorporating the active compounds in the required amount in an appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation
• dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above
• the preferred methods of preparation are vacuum drying and freeze-drying techniques that yield a powder of the active ingredient plus any additional desired ingredient from previously sterile- filtered solution thereof
• Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets
• liposomal or protemoid encapsulation may be used to formulate the present compositions (as, for example, protemoid microspheres reported in U S Patent No 4,925,673)
• Liposomal encapsulation may be used and the liposomes may be denvatised with various polymers (E g , U S Patent No 5,013,556)
• a description of possible solid dosage forms for the therapeutic is given by Marshall, in Modern Pharmaceutics, Chapter 10, Banker and Rhodes ed , (1979), herein incorporated by reference
• the formulation will include the compounds described as part of the invention (or a chemically modified form thereof), and inert ingredients which allow for protection against the stomach environment, and release of the biologically active
• the location of release may be the stomach, the small intestine (the duodenum, the jejunem, or the ileum), or the large intestine
• the release will avoid the deleterious effects of the stomach environment, either by protection of the composition or by release of the compounds beyond the stomach environment, such as in the intestine
• a coating impermeable to at least pH 5 0 is essential
• examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate tnmellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquatenc, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac
• a coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach This can include sugar coatings, or coatings that make the tablet easier to swallow
• Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic / e powder for liquid forms, a soft gelatin shell may be used
• the shell material of cachets could be thick starch or other edible paper
• moist massing techniques can be used
• the therapeutic can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1mm
• the formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets
• the therapeutic could be prepared by compression
• Colourants and flavoring agents may all be included
• compounds may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents
• diluents could include carbohydrates, especially mannitol, alpha-lactose, anhydrous lactose cellulose, sucrose, modified dextrans and starch Certain inorganic salts may be also be used as fillers including calcium tnphosphate, magnesium carbonate and sodium chloride Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell
• Disintegrants may be included in the formulation of the therapeutic into a solid dosage form
• Materials used as disintegrants include but are not limited to starch including the commercial disintegrant based on starch, Explotab Sodium starch glycolate Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium algmate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used
• Another form of the disintegrants are the insoluble cationic exchange resins
• Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth Algmic acid and its sodium salt are also useful as disintegrants
• Binders may be used to hold the therapeutic compounds together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin Others include methylcellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.
• MC methylcellulose
• EC ethyl cellulose
• CMC carboxymethyl cellulose
• PVP polyvinyl pyrrolidone
• HPMC hydroxypropylmethyl cellulose
• Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to: stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, and Carbowax 4000 and 6000.
• the glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.
• a surfactant might be added as a wetting agent.
• Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
• anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
• Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride.
• nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the compounds either alone or as a mixture in different ratios.
• Additives which potentially enhance uptake of the compounds are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.
• Controlled release formulation may be desirable.
• the compounds could be incorporated into an inert matrix that permits release by either diffusion or leaching mechanisms i.e., gums.
• Slowly degenerating matrices may also be incorporated into the formulation.
• Another form of a controlled release of this therapeutic is by a method based on the Oros therapeutic system (Alza Corp.), i.e. the drug is enclosed m a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects.
• Some enteric coatings also have a delayed release effect
• Film coating may be carried out in a pan coater or in a fluidized bed or by compression coating
• pulmonary delivery of the compounds may be delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream
• Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered-dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art
• Ultravent nebulizer manufactured by Mallinckrodt, Inc., St. Louis Missouri
• Acorn II nebulizer manufactured by Marquest Medical Products, Englewood, Colorado
• the Ventolm metered dose inhaler manufactured by Glaxo Inc Research Triangle Park, North Carolina
• the Spmhaler powder inhaler manufactured by Fisons Corp , Bedford, Massachusetts
• each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and/or carriers useful in therapy Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated
• Formulations suitable for use with a nebulizer will typically comprise the compounds suspended in water
• the formulation may also include a buffer and a simple sugar (e g , for protein stabilization and regulation of osmotic pressure)
• the nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the compounds caused by atomization of the solution in forming the aerosol
• Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the compounds suspended in a propellant with the aid of a surfactant
• the propellant may be any conventional material employed for this purpose such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon or a hydrocarbon, including t ⁇ chlorofluoromethane, dichlorod fluoromethane, dichlorotetrafluoroethanol, and 1 ,1 ,1 ,2-tetrafluoroethane, or combinations thereof
• Suitable surfactants include sorbitan tnoleate and soya lecithin Oleic acid may also be useful as a surfactant
• Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing the compound and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e g , 50 to 90% by weight of the formulation
• a bulking agent such as lactose, sorbitol, sucrose, or mannitol
• the compounds (or derivative) should most advantageously be prepared in particulate form with an average particle size of less than 10 microns, most preferably 0 5 to 5 microns, for most effective delivery to the distal lung
• Nasal delivery of the compounds is also contemplated Nasal delivery allows the passage of the protein to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung
• Formulations for nasal delivery include those with dextran or cyclodextran
• Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier
• the specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved and (b) the limitations inherent in the art of compounding such an active material for the treatment sought
• the quantity of active compound to be administered will be largely dependent on, the toxicity and specific activity of compound, the subject to be treated and the degree of treatment required. Precise amounts of compound required to be administered may depend on the judgement of the practitioner and may be peculiar to each subject.
• composition may be given as a single dose schedule, or preferably, in a multiple dose schedule.
• a multiple dose schedule is one in which a primary course of delivery may be with 1 to 10 separate doses, followed by other doses given at subsequent time intervals required to maintain or reinforce the treatment.
• the dosage regimen will also, at least in part, be determined by the need of the individual and the judgement of the practitioner.
• composition of the present invention containing the non-peptidyl compounds may be administered in conjunction with other agents and compounds, for example, immunoglobulins, sulfonylureas, biguanides, ⁇ - glucosidase inhibitors, thiazolidinediones, diazoxide, octreotide, insulin secretogogues as appropriate.
• agents and compounds for example, immunoglobulins, sulfonylureas, biguanides, ⁇ - glucosidase inhibitors, thiazolidinediones, diazoxide, octreotide, insulin secretogogues as appropriate.
• a method for identifying a non- peptidyl compound possessing ionic and hydrophobic chemical moieties spatially located so as to mimic particular ionic and hydrophobic amino acid residues of insulin which are associated with the binding of insulin to its receptor comprising the steps of: (1) comparing the three dimensional structure of the non- peptidyl compound with a three dimensional pharmacophore of an active site of insulin; and (2) selecting a non-peptidyl compound with ionic and hydrophobic chemical moieties spatially located so as to mimic said site.
• the active site comprises at least four amino acid residues selected from the group A21 Asn, B21 Glu, A17 Glu, B24 Phe, B25 Phe, A19 Tyr, B12 Val, B16 Tyr, A2 lie, A3 Val and A1 Gly, with at least one amino acid residue being selected from the group A21 Asn, B21 Glu and A17 Glu.
• the active site comprises one of the following groups of amino acid residues:-
• Comparison of the three dimensional structure of the non-peptidyl compound with the three dimensional pharmacophore may involve comparison of a minimum energy structure of the non-peptidyl compound with a three dimensional pharmacophore of the active site of insulin, the position of the ammo acid residues comprising the active site being determined with reference to crystal and/or NMR solution structures of insulin and its analogues
• the three dimensional pharmacophore may incorporate tolerance spheres about the positions of the respective am o acids to reflect the conformational flexibility of insulin
• the positions of the am o acid residues of the active site are defined relative to one another by the following three dimensional coordinates (in hundredths of Angstroms) within tolerance spheres of between 1 5 and 5
• a B12 Val 343.9, 157 2, -242 6
• B25 Phe 113, -5 3, 780 8
• B24 Phe 92 9, 569 7, 43 2
• B16 Tyr (361 8, 1068, -467 5)
• B21 Glu (-84 9, 1711 2, 99 6)
• A17 Glu -1079 2, 1005.6, - 67 9
• A19 Tyr 763 8, 49 2, -4 1)
• A21 Asn (-344 2, 908, 677 9) A1 Gly (-870.2, - 574 9, 127 9), A2 He (-489 8, -30, -218 1 ), and A3 Val (-237 6, -703 3, -348 4)
• the tolerance spheres for each residue are as follows 5A for residues B24 Phe, B25 Phe, A17 Glu, B21 Glu, A21 Glu, A1 Gly, A2 He, 3 6 A for residues A19 Tyr, B16 Tyr, B12 Val and 3A for A3 Val
• An efficient means to select a non-peptidyl insulin mimetic compound from a potentially large number of non-peptidyl compounds involves comparing non- peptidyl compounds against a three dimensional pharmacophore of insulin using a computer program, for example Catalyst (MSI), to screen one or more computerised databases of three dimensional chemical structures of non-peptidyl compounds
• MSI Catalyst
• the am o acids in the active site are preferably represented by the following Catalyst defined features B12 Val - hydrophobic B25 Phe - hydrophobic, B24 Phe - hydrophobic, B16 Tyr - hydrophobic B21 Glu - negative lonisable, A17 Glu - negative lonisable, A19 Tyr - hydrophobic, A21 Asn - negative lonisable, A1 Gly - positive lonisable, A2 lie - hydrophobic, and A3 Val - hydrophobic
• ammo acid residues represented within Catalyst as hydrophobic may be further specified as B12 Val - aliphatic, B25 Phe - aromatic, B24 Phe - aromatic, B16 Tyr - aromatic, A19 Tyr - aromatic, A2 He - aliphatic, and A3 Val - aliphatic
• a method for determining whether a non-peptidyl compound identified by the third embodiment of the invention is an agonist or an antagonist comprising the step of exposing the compound to an insulin or insulin like receptor and measuring the change in biological activity following exposure of the compound to the receptor
• the NMR solution structure pdbl hiu ent was chosen from the Brookhaven protein database to allow for flexibility of the insulin molecule in solution Additionally, because the insulin crystal structure is reported to be an inactive "closed” conformation and that an "open" conformation, represented by glyc ⁇ ne[B24] insulin, is active, the NMR solution structure, pdbl hit, of Gly[B24] ⁇ nsul ⁇ n was also chosen for pharmacophore construction
• the pdbl hiu ent file contained a collection of eleven models (MODEL 1 through MODEL 1 1 ) while the glycine mutant contained nine models (MODEL 0 through MODEL 8). Each structure represented a possible solution conformation of the molecule that satisfies the NMR distance data.
• the eleven native insulin models (HIU 1-11 ) were overlaid with each other and an average structure was determined. This structure was processed by fixing the alpha carbons and subjecting it to 100 steps of Steepest Descent minimisation (using CHARM force field) to normalise the slightly distorted coordinates of the rest of the molecule. This same procedure could not be used with the glycine analog due to the larger variation among the structures; therefore each model was extracted as a separate molecular structure file. Since the pharmacophore is based upon the native structure of insulin, each glycine model was re-mutated back to Phenylalanine at position B24 using QUANTA'S Protein Design facility.
• the conformation of the phenylalanine side chain was adjusted to minimise bad contacts and ' the side chain rotamer most likely to be found by Karplus rules was assigned.
• the structures were then analysed for their degree of "openness” by measuring the distance between a pseudo atom (placed between residues A2 and A3) and residue B25.
• the average native insulin structure and two glycine mutant structures HIT3 and HIT4 were chosen to represent a range of "openness' in insulin conformations.
• hypotheses were then used to rapidly screen chemical databases - that contained representative three-dimensional structures of each compounds likely minimum energy conformations, for those compounds that contained specific functional groups that could be positioned in the tolerance spheres of each hypothesis.
• Hypotheses 2 and 7a with the tolerances about each of the features of the hypotheses (negative ionisable, positive ionisable and hydrophobic) as listed in Figure 3 identified the following compounds:-
• IM 175 was identified by a number of hypotheses. The number of hypotheses that identified IM 175 depended upon the tolerances of features within each hypothesis. Figure 4 shows the multiple hypotheses that identified compound IM 175 and the tolerances of features in each hypothesis.
• the Hepes buffer system used to dilute the compound depended on the source of insulin receptor preparation used for each assay
• each of the compounds was evaluated in three assays of insulin action 1 ) Insulin receptor autophosphorylation, 2) Phosphorylation of a synthetic substrate specific for insulin receptor (PTK assay) and 3) glucose uptake by 3T3L1 adipocyte cells Insulin Receptor Phosphorylation
• Insulin receptor phosphorylation was assessed using a modification of the method described by Li et al. (21).
• Insulin receptors prepared from CHO cells stably expressing full-length insulin receptors (CHO.T11 ) (15 ⁇ L) were preincubated at room temperature for 20 minutes with 7.5 ⁇ L of 50 mM HEPES, pH 7.4 containing 150 mM NaCl, 0.1 mM phenylmethylsulfonylfluoride, 10 mM MgCI2, 2 mM MnCI2 and 0.1 % Triton X-100 and varying concentrations of human insulin or putative insulin mimetic compound. After this preincubation, 2.5 ⁇ L of 1 mM ATP, 100 mM
• Protein tyrosine kinase activity was determined using a modification of the method of N Konstantopoulos (22) The assay involved measuring the effect of
• the assay was commenced by adding 5 ⁇ L of an insulin receptor solution in 50 mM Hepes, pH 7 5 to 5 ⁇ L of the test compound in 50 mM Hepes, pH 7 5, 5 ⁇ L of FYF peptide in 40 mM imidazole pH 7 3 containing 40 mM ⁇ -glycerophosphate, 1 mM EGTA 100 mM MgCl2, 5 mM MnCl2 and 0 05% BSA, 5 ⁇ L of phosphorylation mixture containing 0 1 mM ATP, 0 5 mM sodium vanadate and
• Glucose transport was determined using a modification of the method of S J Isakoff et al (23) The assay involved measuring the effect of compounds on the
• the cell line, 3T3-L1 derived from mouse embryo fibroblasts, was grown in 250 L tissue culture flasks containing 15 mL medium made up of DMEM (Dulbecco's modified Eagle's medium containing glutamine, 2 mM, sodium bicarbonate, 30 mM penicillin 71 5 mg/litre streptomycin, 152 5 mg/ tre) supplemented with 10% foetal calf serum (FCS) pH 7 4
• DMEM Dulbecco's modified Eagle's medium containing glutamine, 2 mM, sodium bicarbonate, 30 mM penicillin 71 5 mg/litre streptomycin, 152 5 mg/ tre
• FCS foetal calf serum
• 3T3-L1 fibroblast cells from one tissue culture flask were suspended in 50 mL of DMEM media containing 10% FCS and 0 5 mL of this suspension were seeded into each well of two 48-well tissue culture plates The differentiation of the cells was initiated when the fibroblast cells were fully confluent Routinely, differentiation was induced on cells that had been growing in tissue culture plates for 4-5 days Cells were exposed to 0 5 mL of DMEM medium containing 10% FCS, 2 ⁇ g/mL insulin, 0 1 ⁇ g/mL dexamethasone, 0 5 mM isobutylmethylxanthine and 1 ug/mL biotin for three days Cells were then incubated a further three days in 1 mL of post differentiation DMEM medium containing 10% FCS and 2 ⁇ g/mL insulin The cells were subsequently maintained with DMEM medium containing 10% FCS The differentiated cells were examined microscopically between 11 and 13 days after differentiation to ensure they displayed the a
• NIH 3T3 HIR3 5 cells which is 30 fold lower than that reported for receptor phosphorylation They also studied a number of insulin mimetic monoclonal anti- msulin receptor antibodies and showed differential stimulation of 2-deoxyglucose uptake in NIH 3T3 HIR3 5 cells (100%), [ 3 H]thym ⁇ d ⁇ ne incorporation (30%) and 32 P incorporation into insulin receptor ⁇ -subunit (0%) However, a later publication showed that in using a more sensitive assay for receptor autophosphorylation, the same antibodies stimulate phosphorylation of the insulin receptor ⁇ -subunit (25) The differences between assays is not clearly understood but may be due in part to differences in temperature and incubation times of the experiments (24) However, it is also recognised that there are substantial differences between whole cell and broken cell preparations (25) For these reasons it is necessary to characterise each compound in a number of assays
• mice Six week old male CD-1 mice were injected intra pentoneally with a sterile solution of streptozotocm prepared in 100 mM citrate buffer, pH 4 65 at a dose of 200 mg/kg and an injection volume of 16 7 mL/kg Mice were housed in a 12 h light/dark cycle and fed a normal chow diet ad libitum Drinking water was supplemented with 5% sucrose for the first two days following streptozotocm injection before being replaced with tap water thereafter After 4 days, blood was taken from the saphenous vein of the mice using the method of Hem, A et al (26) Random non-fasting blood glucose levels were determined on venous blood using an tended glucometer (Bayer) Mice with blood glucose levels of greater than 15 mmol/L were characterised as diabetic Blood Glucose Level Measurement
• mice Blood glucose levels of mice were determined, followed by an intra peritoneal dose of IM compound dissolved in diluent (1 6% glycerin, 0 25% m-cresol, pH 6 75) Mice were then bled periodically and whole blood glucose levels were determine using a one-touch tended glucometer (Bayer) The ability of compounds to lower blood glucose levels was compared at each time point to mice injected with diluent alone (negative control) and those injected with a 2U/kg intra peritoneal dose of human insulin (positive control)
• IM 129 shows experimental data characteristic of the other IM compounds listed above These characteristics are detailed below All other IM antagonist compounds are included thereafter with minimum discussion, unless explanation of an extraordinary result is required
• This compound may be synthesised by the method described by S W Djuric et al (27), incorporated herein by reference
• IM 129 competes with insulin for binding to a number of receptor sources
• Displacement plots from competition studies conducted using membrane-bound receptor preparations display a characteristic skewed bell-shaped isotherm with a peak in I insulin binding at about 100 to 200 ⁇ M
• IM 129 inhibits insulin binding with an apparent Ki between 320 ⁇ M and 550 ⁇ M
• Figure 6 A typical displacement plot from four separate experiments can be seen in Figure 6
• Purified receptor preparations yield a less complicated displacement plot with an estimated Ki of 189 ⁇ M (LIGAND 1-s ⁇ te fit), as can be seen in Figure 7 which is consistent with observed differences in insulin binding to membrane- associated and soluble receptor preparations described in the background art
• IM 129 displaces I IGF-1 binding from human placental plasma membranes (HPPM) in a dose dependent manner with an apparent Ki of 0 8-1 3 mM, as illustrated by Figure 11 This is approximately two fold higher than the apparent
• IM 025 competes with insulin binding to insulin receptors from a number of sources with an estimated Ki of 100 - 400 ⁇ M, as seen in Figures 12 to 14 Figure
• 125 12 shows a typical competition experiment of IM 025 on I insulin binding to human placental plasma membranes
• the calculated Ki over 5 experiments is 335 uM ⁇ 34 ⁇ M
• Figure 13 shows a typical competition experiment of IM 025 on
• IM 025 is an antagonist of insulin action IM
• Figure 17 shows that IM 071 competes for insulin binding to HPPM in a dose dependent manner, with an inhibition constant (calculated for two experiments) of 186 ⁇ M ⁇ 33 ⁇ M IM 071 is structurally related to IM 129 and is likely to have similar antagonistic effects of insulin in biological assays
• IM 127 displays a skewed bell-shaped displacement plot of I insulin binding to
• IM 127 displays insulin antagonistic activity in a number of assays, summarised in Figures 20 and 21 Figure 21 specifically shows that the estimated Ki is 60 ⁇ M in the glucose transport assay EXAMPLE 5
• 3,4-Dihydro-8-propyl-7-[[3-[2-ethyl-5-hydroxy-4-(1 H-pyrazol-3- yl)phenoxy]propyl]oxy]-2H-1-benzopyran-2-carboxylic acid can be synthesised by the method described by R. W. Harper et al. (28), incorporated herein by reference.
• Figures 22 and 23 illustrate that IM 132 effects insulin binding with an apparent inhibition constant of 200 to 450 ⁇ M, which is dependent upon receptor source.
• IM 132 is an antagonist of insulin action because it does not increase 32p incorporation into FYF peptide or activate glucose transport.
• Figures 24 and 25 illustrate that it also inhibits the insulin stimulation of these assays.
• the estimated inhibition constant of the compound in the PTK and glucose transport assays is 360 ⁇ M and 64 ⁇ M respectively.
• IM 132 shows significant synergism with insulin in the glucose transport assay, increasing the effect of a submaximal dose of insulin (2 nM) to levels above those attained by 100 nM insulin. This indicates that IM 132 may be interacting with the insulin receptor at the insulin binding site in a more complex manner than IM 129.
• Figure 26 shows that IM 134 competes with I insulin binding to HPPM with an apparent Ki of 900 ⁇ M.
• IM 134 is an antagonist of insulin action because it does not increase P incorporation into FYF peptide or glucose transport and inhibits the insulin stimulation of these assays, as can be seen in Figures 27 and 28.
• the estimated inhibition constants of the compound in these assays are 300-400 ⁇ M and 60-110 ⁇ M respectively.
• IM 143 competes with I insulin binding to HPPM with an apparent Ki of about 2 mM.
• IM 143 is an antagonist of insulin action because it does not stimulate glucose transport activity and inhibits a stimulating (2 nM) dose of insulin with an estimated inhibition constant of 40 ⁇ M as illustrated in Figure 30.
• IM 144 causes a skewed bell-shaped displacement plot of I insulin binding to HPPM with an apparent Ki of 900 ⁇ M
• IM 144 is an antagonist of insulin action because it does not increase P incorporation into FYF peptide or activate glucose transport and inhibits the insulin stimulation of these assays, as shown in Figures 33 and 34
• the estimated inhibition constant of the compound in the PTK and glucose transport assays is 130 ⁇ M and 125 ⁇ M respectively
• IM 145 competes for I insulin binding to HPPM with an apparent Ki of about 1000 ⁇ M as shown in Figure 35
• FIGS 36 and 37 show that IM 145 is an antagonist of insulin action because it does not stimulate PTK or glucose transport activity and inhibits a stimulating dose of insulin (400 nM and 2 nM respectively) in each assay with an estimated IC50 of 90 and 60 uM respectively EXAMPLE 10
• 8-Propyl-7-[3-[4-(4-fluorophenyl)-2-ethyl-5-hydroxyphenoxy]propoxy]-3,4-dihydro- 2H-1-benzopyran-2-carboxylic acid can be obtained by the method described in International Application WO 95/17183 (Eli Lilly and Co) incorporated herein by reference.
• Figure 38 shows that IM 171 competes for I insulin binding to HPPM with an apparent Ki of 370 ⁇ M.
• IM 171 is an antagonist of insulin action. It does not increase P incorporation into FYF peptide or activate glucose transport and inhibits insulin stimulation of each of these assays, as is evident from Figures 39 and 40.
• the estimated IC50 in the PTK and glucose transport assays are 250 ⁇ M and 80 ⁇ M respectively.
• IM 172 competes for I insulin binding to HPPM with an apparent Ki of 218 ⁇ M, as Figure 41 illustrates.
• IM 172 is an antagonist of insulin action. It does not increase P incorporation into FYF peptide or the insulin receptor ⁇ -subunit and does not activate glucose transport in 3T3L1 cells. As Figures 42 to 44 demonstrate, it also inhibits the insulin stimulation of each of these assays.
• the estimated inhibition constant of the compound is 80 ⁇ M in the both the PTK and glucose transport assays.
• IM 103 have been classified as agonists of insulin action.
• IM 140 causes an increase in I insulin binding with
• I insulin binding isotherm can be explained by a similar mechanism as described for IM 129.
• IM 140 is likely to crosslink the ⁇ 1 and ⁇ 2 sites of adjacent ⁇ -subunits, because it activates the insulin receptor.
• IM 140 is also synergistic with insulin, indicating that both insulin and IM 140 interact with the receptor in a similar
• IM 140 is an agonist of insulin action because, as Figures 47 to 49 illustrate, it activates glucose transport in 3T3L1 cells, as Figures 51 and 52 illustrate, it causes phosphorylation of an exogenous insulin receptor tyrosine kinase substrate by insulin receptors of differing levels of purity and, as Figure 53 demonstrates, lowers blood glucose levels in experimentally induced diabetic mice.
• IM 140 has an efficacy of 25-40% compared with insulin (inter experimental variation) and exhibits a bi-phasic dose-response curve with an apparent EC50 of 20-30 ⁇ M (Glucose transport).
• IM 140 acts in a synergistic manner with a sub-maximal dose of insulin (2 nM) to stimulate glucose transport to 80% of maximal efficacy (100 nM insulin) at a concentration of 15 ⁇ M with an apparent EC50 of 8 ⁇ M.
• the observed difference in concentration of IM 140 effecting insulin binding and biological activity appears to be, at least in part, an artefact of inter-assay variation.
• IM 140 was found to effect each assay over a similar concentration range, as illustrated in Figure 49.
• Figures 51 shows that IM 140 also promotes the ATP-dependent phosphorylation of endogenous FYF peptide in a dose dependent manner.
• Figure 52 indicates that when immunoprecipitated WGA.IR receptors are used as the source of tyrosine kinase activity, IM 140 promotes phosphorylation of endogenous FYF peptide in a bi-phasic manner.
• the immunoprecipitated WGA.IR receptors were only stimulated by a 100 fold excess of IGF-1 relative to insulin, indicating that the preparation was IGF-1 receptor free. This indicates that the phosphorylation of exogenous FYF peptide substrate was via the insulin receptor.
• Figure 53 demonstrates that IM140 significantly lowers blood glucose levels in diabetic mice with an efficacy of about 25% of human insulin at a dose of 35 ⁇ mol/kg. Preliminary studies also indicate that IM 140 does not decrease blood glucose levels in diabetic mice at a concentration of 100 ⁇ mol/kg. Therefore IM 140 displays a bi-phasic dose response curve in vivo with a peak activity of 20-40 ⁇ mol/kg and an EC50 of 15 ⁇ mol/kg.
• the bi-phasic dose response curve is analogous to growth hormone activation of its receptor. This indicates that IM 140 may be activating the insulin receptor by cross-linking its ⁇ -subunits. This is consistent with the model of insulin's interaction with its receptor proposed by P. De Meyts (14) and L. Schaffer (15). They propose that insulin activates the insulin receptor by cross-linking the ⁇ 1 and ⁇ 2 sites on adjacent insulin receptor ⁇ -subunits. IM 140 presumably contains some features that also enable it to cross-link these sites. The bell shaped dose response curve can then be explained by high concentrations of IM 140 binding each of the ⁇ -subunits thereby preventing cross-linking of the receptor ( Figure 50).
• IM compounds that are antagonists would lack some of the features of IM 140 or due to size or conformational restraints may be unable to interact concomitantly with both the ⁇ 1 and ⁇ 2 sites.
• Antagonist compounds may be similar to growth hormone mutants that are modified at one of their receptor contact sites which leads to them becoming potent antagonists of hGH.
• IM 140 effects I insulin and I IGF-1 binding to HPPM in a dose dependent and complex manner, as can be seen in Figure 54. However, over the range of
• the compound does not compete for I IGF-1 binding to HPPM.
• IM 140 promotes I IGF-1 binding to HPPM which may be a shift to the right of the bell shaped displacement curve observed for I insulin binding.
• Figure 55 indicates that IM140 does not compete for I IGF-1 binding in WGA purified receptor preparations from solubilised CHO wild- type cells. These cells are almost devoid of insulin receptors but have high endogenous populations of IGF-1 receptors.
• IM 140 effects both IGF-1 binding and Insulin binding to membrane preparations considering the homology and degree of cross-reactivity between the hormones and their receptors. Each receptor probably has a common shaped binding domain that can accommodate either hormone in a structurally equivalent manner This result suggests that IM 140 has features that allow it to interact more specifically with the insulin receptor than the IGF-1 receptor within a common binding domain
• IM 175 competes with I insulin for binding to HPPM Figure 56 shows that the apparent Ki is 46-48 ⁇ M IM 175 competes for binding according to a simple 1- site model and does not display the complex binding kinetics associated with other agonist compounds (eg IM 140) This may be because IM 175 is a larger compound and its size may prevent additional molecules of insulin interacting with
• IM 175 promotes glucose transport to levels approaching maximal stimulation by insulin in 3T3L1 adipocyte cells
• Figure 58 shows a typical experiment, the apparent EC50 is about 250 ⁇ M
• Figure 59 demonstrates that IM 175 stimulates autophosphorylation of insulin receptor ⁇ -subunits with about 65% of the efficacy of a 100 nM dose of insulin
• the immunoprecipitated insulin receptors used for this experiment are free of contaminating IGF-1 receptors indicating that IM 175 specifically stimulates the phosphorylation of the insulin receptor
• Figure 60 illustrates that IM 175 can also stimulate the IGF-1 receptor, where immunoprecipitated IGF-1 receptors are free of insulin receptors
• the dose-response curves indicate that IM175 has a lower potency on IGF-1 receptors than it does on insulin receptors
• IM 175 is structurally a symmetrical molecule and is a dimer of other IM compounds It fits a general formula of ZXYXA-AXYXZ whereas IM 140 fits the general formula AXYXZ
• This dime ⁇ c structure may be related to it having the highest efficacy of agonist compounds as other studies have implicated a role of symmetry and dimer structure in increased molecule activity
• small peptide agonists of the erythropoietin receptor and thrombopoeitin receptor when dimensed, activate their respective receptors with greater potency than monomers
• bivalent anti insulin receptor antibodies activate insulin receptors, whereas monovalent Fab fragments do not
• IM 103 competes with insulin for binding to insulin receptors in a dose dependent manner
• the displacement plot is similar to other IM compounds (exemplified by IM 129 and IM 140) and does not fit a simple 1 -site or 2-s ⁇ te model
• the apparent inhibition constant is dependent upon receptor source and is 137 ⁇ 45 ⁇ M (LIGAND, 1-s ⁇ te fit) in HPPM, 132 ⁇ 29 ⁇ M in CHO T11 cells and 83 ⁇ M in WGA IR receptors, as can be seen in Figures 61 , 62 and 63 respectively
• IM 103 is an agonist of insulin action displaying a bi-phasic biological dose response curve with an apex at concentration of 110 ⁇ M and an apparent EC50 of 45 ⁇ 7 ⁇ M, as illustrated by Figure 64
• the efficacy is about 15% of that achieved at a maximal dose of insulin (100 nM) IM 103 abolishes the insulin stimulation of glucose transport in a dose dependent manner
• the dose response curve indicates complex kinetics Specificity
• IM 103 displaces I IGF-1 binding from HPPM in a dose dependent manner with an apparent Ki of 59 ⁇ M (LIGAND estimated 1 -site fit), as can be seen in Figure
• IM 103 appears to have a higher specificity for IGF-1 receptors than insulin receptors.

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