Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
  • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor

Definitions

• This disclosure relates to methods of screening agents for their ability to restore functionality to a mutated gonadotropin-releasing hormone receptor, increase the amount of wild-type GnRHR on the cell surface, or both, and methods of using identified agents to treat a subject having hypogonadism.
• BACKGROUND Disease-causing receptor mutations were widely believed to lose function as a result of inability to engage in receptor-ligand or receptor-effector binding interactions.
• receptor misfolding and subsequent misrouting is a mechanism that results in loss of receptor function (Janovick et al., J. Gin. Endocrinol. Metab., 87:3255-62, 2002; Leanos-Miranda et al, J. Gin. Endo. Metab., 87:4825-8, 2002; Conn et al, Molecidar Interventions, 2:308-16, 2002).
• Gonadotropin-releasing hormone (GnRH) plays a central role in neural regulation of reproductive function.
• GnRH gonadotropin-releasing hormone receptor
• GnRHR gonadotropin-releasing hormone receptor
• Sequence analysis of the GnRHR is consistent with the seven transmembrane domain motif, characteristic of the G protein-coupled receptors superfamily (Ulloa-Aguirre and Conn. G protein-coupled receptors and the G protein family. In: Handbook of Physiology- Endocrinology.
• HH congenital hypogonadotropic hypogonadism
• GnRHR mutational defects in GnRHR.
• At least 14 mutations of GnRHR are associated with HH.
• Mutant E 90 K has been 'rescued' by deleting K 191 (which, when present, decreases expression of hGnRHR at the plasma membrane) or by adding a C-terminal sequence (Maya-Nunez et al, J. Clinical Endocrinol. Metab. 87:2144-9, 2002).
• previous approaches to correct defective receptors include genetic approaches, such as increased receptor expression to produce larger numbers of receptors (Cheng et al, Am. J. Physiol 268:L615-24, 1995). However, rescue by these genetic approaches is not presently practical for in vivo use.
• GnRHR gonadotropin-releasing hormone receptor
• agents from at least three different chemical classes indoles, quinolones, and macrolides
• GnRHR gonadotropin-releasing hormone receptor
• a model is proposed whereby chemically distinct agents serve as molecular scaffolding, cause mutant GnRH receptors to fold correctly and thereby avoid detection by the cellular quality control apparatuses. The correctly folded receptor can then traffic to the cell membrane, and be available for ligand binding.
• the agents that were successful in restoring function to one GnRHR mutant usually rescued all mutants (that could be rescued by any of the peptidomimetics). However, function was not restored to particular GnRHR mutants (human: S R and S 217 R; rat des 260"265 -GnRHR and C 229 A-GnRHR) by any agent, indicating that receptors these were either grossly deformed or that loss-of-function was due to inability to bind ligand.
• the efficacy of each agent was proportional to the binding affinity of the molecules for the WT receptor.
• agents that restore function to a mutant GnRHR are GnRHR antagonists, and therefore may compete with the natural ligand. Such agents are believed to stabilize the ligand binding site of the mutant receptors. In another example, agents that restore function to a mutant GnRHR do not compete for the natural ligand binding site. Therapeutic agents that bind outside the natural ligand binding site of GnRHR do not interfere with subsequence activation of the receptor with an agonist.
• the disclosed pharmacoperones have one or more of the following characteristics: specificity for the receptor being rescued, ability to arrive at the correct cellular locus (enter the cell, get to the endoplasmic reticulum, and remain stable long enough to bind the nascent molecule), and ability to dissociate from the molecule (or, in the alternative, not compete with the physiological ligand) after it arrives at the appropriate target locus (such as the cell surface).
• the methods include contacting the agent with a mutant GnRHR under conditions that allow interaction between the agent and the receptor, and then determining whether functionality was restored to the receptor.
• determining whether functionality was restored to the mutant GnRHR by the agent for example determining an amount of inositol phosphate (IP) production, determining an amount GnRHR agonist binding, determining an amount of surface-bound mutant- GnRHR-ligand complex internalized, or combinations thereof.
• IP inositol phosphate
• the screening methods include contacting a GnRHR antagonist with GnRHR under conditions that allow interaction between the agent and the receptor, and screening the antagonist for ability to restore GnRHR function.
• the inhibitory concentration (IC 50 ) of the agent is determined.
• Agents having a particular ICso such as indole or quinolone derivatives having an IC 50 of less than 5 nM (for example less than 3 nM, less than 2.5 nM, or less than 2 nM), or macrolide derivatives having an IC 50 of less than 700 nM (for example less than 100 nM, less than 20 nM, or less than 2 nM), are selected for further study.
• methods for restoring function to a mutant GnRHR by contacting the identified agents, such as an indole-, quinilone-, or macrolide-derivative, with the mutant receptor.
• a therapeutically effective amount of such an agent is used to treat a subject having a mutant GnRHR, for example a subject having HH.
• the inventor has also determined that normal expression of human GnRHR, but not rat GnRHR, was increased by the agents that were successful in rescuing mutant GnRHR.
• the presence of Lys 191 and other features (Janovick et al, Endocrine 22:317- 28, 2003) in the primate sequence limits the percentage of the synthesized receptor that reaches the plasma membrane to about 50%.
• agents that can restore function to a mutant GnRHR can also be used to increase expression of wild- type human GnRHR at the plasma membrane.
• FIGS. 1-3 are graphs showing the efficacy (assessed by IP production) of each of (1) indoles, (2) quinolones and (3) erythromycin macrolides on restoration of function to the GnRHR mutant E 90 K.
• FIGS. 4-6 are bar graphs showing the effect of 1 ⁇ g/ml of each (4) indole, (5) quinolone and (6) erythromycin macrolide in restoring function to each GnRHR mutant.
• IP production in the presence of 10 "7 M buserelin.
• SEM bars were omitted. The standard deviation was typically less than 10% of the corresponding mean.
• FIG. 7 is a bar graph showing the unrescued (no drug) coupling of receptor in the absence (upper graph) or presence (lower graph) of 10 "7 M buserelin.
• Agent Any polypeptide, compound, small molecule, organic compound, salt, polynucleotide, pharmacologic of biologic chaperone, or other molecule of interest.
• Analog An agent (such as an organic chemical compound) that is structurally similar to another, but differs slightly in composition, for example the replacement of one atom by an atom of a different element or functional group.
• an analog ofIn3, ((2S)-2-[5-[2-(2-azabicyclo[2.2.2]oct-2-yl)-l,l-dimethyl-2-oxoethyl]-2-(3,5- dimethylphenyl)- lH-indol-3 -yl] -N-(2-pyridin-4-ylethyl)propan- 1 -amine, is structurally similar to In3, and has a similar effect on restoring function to a GnR ⁇ R mutant such as E 90 K.
• Binding A specific interaction between two molecules, such that the two molecules interact. Binding can be specific and selective, so that one molecule is bound preferentially when compared to another molecule. In one example, specific binding is identified by a disassociation constant (E ). In another example, specific binding of an antagonist for a receptor is identified by an inhibitory concentration (IC 5 0).
• E disassociation constant
• IC 5 0 inhibitory concentration
• G protein-coupled receptor A superfamily of proteins, characterized by seven transmembrane alpha-helices, that signal through interaction with a family of heterotrimeric GTP-binding proteins, referred to as G proteins. Examples include, but are not limited to, beta-adrenergic receptor (betaAR), cystic fibrosis transmembrane conductance regulator (CFTR), gonadotropin-releasing hormone receptor (GnR ⁇ R), rhodopsin, and vasopressin receptor (V2R).
• betaAR beta-adrenergic receptor
• CFTR cystic fibrosis transmembrane conductance regulator
• GnR ⁇ R gonadotropin-releasing hormone receptor
• V2R vasopressin receptor
• GnR ⁇ R Gonadotropin releasing hormone receptor
• GnR ⁇ receptors belong to the family of G protein-coupled receptor proteins and have been localized to the anterior pituitary, brain and reproductive organs as well as many steroid-dependent rumor tissues.
• the activated GnR ⁇ R-G q /1. 1 protein complex activates the membrane-associated enzyme phospholipase Cp, leading to inositol 1,4,5-trisphosphate (IP) production and the release of intracellular calcium.
• IP inositol 1,4,5-trisphosphate
• GnR ⁇ R includes any GnR ⁇ R gene, cD ⁇ A, R ⁇ A, or protein from any organism and includes a GnR ⁇ R that can normally traffic to the cell surface and bind GnRH.
• wild-type GnRHR nucleic acid sequences include, but are not limited to, GenBank Accession No. AF001950 (human cDNA) and GenBank Accession No. S59525 (rat mRNA).
• GnRHR amino acid sequences include, but are not limited to: Genbank Accession Nos: AAB71348 (human) and AAB2642 (rat).
• a GnRHR sequence includes a full-length wild-type (or native) sequence, as well as GnRHR allelic variants, variants, fragments, homologs or fusion sequences that retain wild-type function.
• GnRHR agonist Agents that can bind to GnRHR and initiate the physiological and pharmacological responses characteristic of GnRHR.
• Examples include native GnRHR ligands, such as GnRH, as well as other agents that can mimic the action of GnRH.
• Particular examples include buserelin (D-tert-butyl-Ser 6 , des-Gly 10 , Pro 9 , ethylamide-GnRH) and leuprolide (D-Leu 6 , Pro 9 , des-Gly 1 °-ethylamide-GnRH).
• GnRHR antagonist Agents that bind to the ligand-binding site of GnRHR and interfere with binding of a ligand or agonist to the GnRHR binding site, thereby resulting in decreased GnRHR-associated responses normally induced by the ligand or agonist.
• the inhibitory activity of an antagonist for a receptor is represented as an inhibitory concentration (IC 50 ), wherein better inhibitors have lower IC 50 values.
• Antagonism can be competitive and reversible (it binds reversibly to a region of the receptor in common with the agonist.) or competitive and irreversible (antagonist binds covalently to the agonist binding site, and no amount of agonist can overcome the inhibition).
• GnRHR ligand Agents that can bind to the ligand-binding site of GnRHR. In some examples, such agents bind reversibly.
• the native GnRHR ligand is GnRH. Includes GnRHR agonists that bind at the ligand-binding site.
• Hypogonadism An underactivity of the sex glands. In men, hypogonadism is a condition that occurs when the testicles do not produce enough testosterone. In women, hypogonadism is a condition that occurs when the ovaries do not produce enough estrogen. Primary hypogonadism occurs when there is a problem with the testicles/ovaries themselves.
• Secondary hypogonadism occurs when there is a problem with the pituitary gland.
• the hypothalamus secretes GnRH to stimulate the pituitary gland.
• the pituitary gland secretes other hormones (follicle stimulating hormone (FSH) and luteinizing hormone (LH)).
• FSH follicle stimulating hormone
• LH luteinizing hormone
• the ovaries female and testes (male) to secrete hormones (estrogen and testosterone, respectively) responsible for normal sexual development in puberty. Any disruption in this cascade causes a deficiency of the sex hormones and halts normal pubertal sexual maturation.
• FSH follicle stimulating hormone
• LH luteinizing hormone
• Any disruption in this cascade causes a deficiency of the sex hormones and halts normal pubertal sexual maturation.
• One example of secondary hypogonadism is hypogonadotropic hypogonadism.
• hypogonadism can occur during fetal development, at puberty, or in adults. Symptoms associated with hypogonadism include: erectile dysfunction in men (the inability to achieve or maintain an erection), infertility, decreased sex drive, decrease in beard and growth of body hair, decrease in size or firmness of the testicles, decrease in muscle mass and increase in body fat, enlarged male breast tissue, hot flashes, mood swings, irritability, depression, fatigue, osteoporosis (decreased bone density), delayed puberty, and combinations thereof. There are several causes of hypogonadism, including: Klinefelter's syndrome
• TRT testosterone replacement therapy
• HRT hormone replacement therapy
• GnRH androgens
• GnRH can also help increase testosterone and estrogen levels.
• the present disclosure provides additional methods that can be used to treat hypogonadism, such as administration of agents that restore function to mutant GnRHR, and administration of agents that increase the presence of wild-type GnRHR on the cell surface.
• hypogonadism include hypotestosteronism (a disorder characterized by lower than normal plasma levels of testosterone) and hypoestrogeneism (a disorder characterized by lower than normal plasma levels of estrogen).
• HH Hypogonadotropic hypogonadism
• KAL-1 associated with X-linked Kallmann Syndrome
• HESX1, LHX3, and PROP-1 pituitary transcription factors
• DAX-1 associated with X-linked adrenal hypoplasia congenital, and SF-1
• leptin, leptin receptor, and prohormone convertase three genes also associated with obesity (leptin, leptin receptor, and prohormone convertase 1).
• Indole (2,3-benzopyrrole) A heterocyclic compound having the chemical formula C 8 H 7 N, and derivatives thereof, such as In30, ((2S)-2-[5-[2-(2- azabicyclo[2.2.2]oct-2-yl)-l,l-dimethyl-2-oxoethyl]-2-(3,5-dimethylphenyl)-lH-indol- 3-yl]-N- ⁇ 2-[4-(methylsulfinyl)phenyl]ethyl ⁇ propan-l-amine); In3 lb, ((2S)-N-[2-(4- carboxyphenyl)ethyl] -2- [5 - [ 1 , 1 -dimethyl-2-(4-methylpiperazin- 1 -yl)-2-oxoethyl] -2- (3,5-dimethylphenyl)-lH-indol-3-yl]propan-l-aminium tri
• an indole includes indole- derivatives that have an IC 50 of about less than 3 nM for hGnRHR.
• Inhibitory concentration is a concentration that inhibits an effect (such as a receptor-mediated effect).
• the amount of inhibitory agent needed to reduce an effect by 50% is an IC 50 .
• the IC50 of a GnRHR antagonist is the concentration of antagonist needed to reduce an amount of ligand binding (such as GnRH) to the receptor by 50%, compared to an amount of ligand binding in the absence of the antagonist. Lower IC 50 values suggest better inhibition.
• Inositol phosphate Includes all phosphorylated states of inositol, such as inositol-1 -phosphate (IPO, inositol-4,5-diphosphate (IP 2 ), inositol- 1,4,5-triphosphate (IP 3 ), and inositol-1, 3, 4,5-triphosphate (IP 4 ).
• IPs are produced following activation of a GnRHR.
• GnRHR for example by binding of a GnRHR ligand or agonist to cell-surface GnRHR, leads to activation of phospholipase C, which catalyzes the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP 2 ) into IP 3 and 1,2-diacylglycerol (DG).
• PIP 2 phosphatidylinositol-4,5-bisphosphate
• IP 3 1,2-diacylglycerol
• Label An agent capable of detection, for example by ELISA, spectrophotometry, flow cytometry, or microscopy.
• a label can be attached to a protein, thereby permitting detection of the protein.
• labels include, but are not limited to, radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).
• Macrolide Any of a large group of antibiotics containing a macrolide ring linked glycosidically to one or more sugars.
• Members of the macrolide antibiotic group include erythromycin, carbomycin, azithromycin, and clarithromycin, and derivates thereof.
• Examples of particular erythromycin-derived macrolides include A-7662.0, (Erythromycin A); A-64755.0 (11-deoxy-l l-[carboxy-phenylethylamino]-6-O-methyl- erythromycin A l l,12-(cyclic carbamate)); A-177775.0, (3'-N-desmethyl-3'-N- cyclopentyl- 11 -deoxy- 11 -[carboxy-(3,4-dichlorophenylethylamino)]-6-O-methyl- erythromycin A 1 l,12-(cyclic carbamate)); and A-222509.0, 3',3'-N-desmethyl-3',3'-N- cyclopropylmethyl- 11 -deoxy- 11 - [carboxy-(3 -chloro,4-fluoro-phenylethylamino)] -6-O- methyl-erythromycin A 1 l,12-(cyclic carb
• a macrolide includes macrolide- derivatives that have an IC 50 of about less than 700 nM for hGnRHR.
• Mammal This term includes both human and non-human mammals. Similarly, the terms “patient,” “subject,” and “individual” includes living multicellular vertebrate organisms, such as human and veterinary subjects.
• Mimetic A molecule (such as an organic chemical compound) that mimics the activity of an agent, such as the activity of a GnRHR antagonist on trafficking of mutant GnRHR to the cell surface.
• Peptidomimetic and organomimetic embodiments are within the scope of this term, whereby the three-dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three- dimensional arrangement of the peptide backbone and component amino acid sidechains in the peptide, resulting in such peptido- and organomimetics of the peptides having substantial specific activity.
• a pharmacophore is an idealized, three-dimensional definition of the structural requirements for biological activity.
• Peptido- and organomimetics can be designed to fit each pharmacophore with computer modeling software (using computer assisted drug design or CADD). See Walters, "Computer-Assisted Modeling of Drugs", in Klegerman & Groves, eds., 1993, Pharmaceutical Biotechnology, Interpharm Press: Buffalo Grove, IL, pp. 165-174 and Principles of Pharmacology (ed. Munson, 1995), chapter 102 for a description of techniques used in computer assisted drug design.
• Mutant GnRHR A GnRHR sequence that includes at least one amino acid substitution or deletion compared to a wild-type sequence, which results in the development of HH in a subject.
• mutant GnRHR examples include, but are not limited to, human N 10 K, T 32 I, E 90 K, Q 106 R, A 129 D, R 139 H, C 200 Y, R 62 Q, L 266 R, C 279 Y, and Y 284 C mutations, as well as rat des325-327, desL237-L241 and C 278 A mutations.
• mutant GnRHR does not include the human S 168 R or S 17 R mutations, or the rat des260-65-GnRHR or C 229 A mutation.
• Peripheral Administration Administration outside of the central nervous system. Peripheral administration does not include direct administration to the brain. Peripheral administration includes, but is not limited to intravascular, intramuscular, subcutaneous, inhalation, oral, rectal, transdermal or intra-nasal administration.
• Quinolone A heterocyclic compound having the chemical formula C 9 H 7 N, and derivatives thereof, such as Q89, (7-chloro-2-oxo-4- ⁇ 2-[(2S)-piperidin-2-yl]ethoxy ⁇ -N- pyrimidin-4-yl-3-(3,4,5-trimethylphenyl)-l,2-dihydroquinoline-6-carboxamide); Q76, (N- (7-chloro-3-(3,5-dimethylphenyl)-2-oxo-4- ⁇ 2-[(2S)- ⁇ i ⁇ eridin-2-yl]ethoxy ⁇ - 1 ,2- dihydroquinolin-6-yl)-N-cyclopropylurea); and Q08, ((2S)-2-(2- ⁇ [7-chloro-6-[(6,7- dimethoxy-3,4-dihydroisoquinolin-2(lH)-yl)carbonyl]-3-(3,5-dimethylphenyl)-2
• quinolone antibiotics such as nalidixic acid, cinoxacin, rosoxacin, and the fluorinated 4-q's. Additional quinolone antibiotics are disclosed in U.S. Patent ⁇ os. 5,990,122; 5,646,163; and 5,385,906, all herein incorporated by reference.
• a quinolone includes quinolone-derivatives that have an IC 50 of about less than 3 nM for hGnRHR.
• Restore function An agent, such as a GnRHR pharmacoperone, is said to restore function to a mutant GnRHR when contact of the agent with a mutant GnRHR results in increased GnRHR-type activity of the mutant receptor, as compared to binding in the absence of the agent. Restoring function to a mutant GnRHR does not require restoration of 100% of wild-type activity. In one example, increasing GnRHR-type activity of the mutant receptor increases binding of a mutant GnRHR to a GnRHR agonist (such as buserelin), as compared to binding in the absence of the agent.
• a GnRHR agonist such as buserelin
• binding of a GnRHR agonist to a mutant GnRHR having restored function increases at least 25% when compared to binding of a mutant GnRHR to GnRHR agonist in the absence of the agent.
• binding of a GnRHR agonist to a mutant GnRHR having restored function increases at least 50%, such as at least 60%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 100%, or even such as at least 200%, when compared to binding of a mutant GnRHR to GnRHR agonist in the absence of an agent.
• Such binding can be performed using the methods disclosed herein (see Example 1).
• an agent such as a GnRHR antagonist
• a GnRHR antagonist is said to restore function to a mutant GnRHR when contact of the agent with a mutant GnRHR results in an increase in GnRH agonist-stimulated inositol phosphate (IP) production, as compared to IP production in the absence of the agent.
• IP GnRH agonist-stimulated inositol phosphate
• IP production by a mutant GnRHR having restored function increases at least 50%, such as at least 60%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 100%, such as at least 200%, or even such as at least 500% when compared to IP production by mutant GnRHR in the absence of the agent.
• IP production can be performed using the methods disclosed herein (see Example 1).
• an agent is said to restore function to a mutant GnRHR when contact of the agent with a mutant GnRHR results in increased surface-bound receptor-ligand complex internalized, as compared to an amount of surface-bound receptor-ligand complex internalized in the absence of the agent.
• surface-bound receptor-ligand complex internalized by a mutant GnRHR having restored function increases at least 25% when compared to IP production by a mutant GnRHR in the absence of the agent.
• surface-bound receptor-ligand complex internalized by a mutant GnRHR having restored function increases at least 50%, such as at least 60%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 100%, or even such as at least 200% when compared to an amount of surface-bound receptor-ligand complex internalized by a mutant GnRHR in the absence of the agent. Determination of surface-bound receptor-ligand complex internalized can be performed using the methods disclosed herein (see Example 2).
• Subject Living multicellular vertebrate organisms, a category that includes, both human and veterinary subjects for example, mammals, rodents, and birds.
• Therapeutically effective amount An amount of an agent (alone or in combination with other therapeutically effective agents) sufficient to achieve a desired biological effect, for example an amount that is effective to increase binding of a GnRHR agonist to a mutant GnRHR by at least a desired amount, increase GnRH agonist-stimulated inositol phosphate (IP) production by a mutant GnRHR by at least a desired amount, increase surface-bound mutant GnRHR-ligand complex internalized by at least a desired amount, or combinations thereof, such as in the cell of a subject to whom it is administered.
• IP inositol phosphate
• it is an amount of an agent effective to increase binding of a GnRHR agonist to a mutant GnRHR by at least a desired amount, such as an increase by at least 25%, at least 50%, at least 75%, at least 20%, or even at least 200% as compared to an amount of binding prior to treatment.
• it is an amount effective to increase GnRH agonist-stimulated IP production by a mutant GnRHR by at least a desired amount, such as increase by at least 25%, at least 50%, at least 75%, al least 20%, or even at least 200% as compared to an amount of IP production prior to treatment.
• it is an amount effective to increase surface-bound mutant GnRHR-ligand complex internalized by at least a desired amount, such as increase by at least 25%, at least 50%, at least 75%, at least 20%, or even at least 200% as compared to an amount of complex internalized prior to treatment. In some examples, it is an amount of an agent that can restore function to a mutant GnRHR (alone or in combination with other therapeutically effective agents) that can improve signs or symptoms of HH due to a GnRHR mutation.
• an agent (alone or in combination with other therapeutically effective agents) that can increase an amount of wild-type GnRHR at the cell surface, for example by at least 10%, at least 20%, or even at least 50% as compared to the absence of the agent.
• a therapeutically effective amount improves signs or symptoms of hypogonadism, for example such a condition associated with decreased functional GnRHR at the cell surface.
• An effective amount of an agent that restores function to a mutant GnRHR, such as a GnRHR antagonist can be administered in a single dose, or in several doses, for example daily, during a course of treatment.
• the effective amount of agent may be dependent on the source of agent administered, the subject being treated, the severity and type of HH being treated, and the manner of administration.
• a therapeutically effective amount of an agent that restores function to a mutant GnRHR can vary from about 1 ⁇ g/kg body weight to about 20 ⁇ g/kg body weight per day, about 1 ⁇ g/kg body weight to about 10 ⁇ g/kg body weight per day, about 10 ⁇ g/kg body weight to about 20 ⁇ g/kg body weight per day, or about 1-2 ⁇ g agent/kg body weight/day.
• a therapeutically effective amount of an agent such as an antagonist, peaks in the serum, then returns to negligible levels indicating that the agent has been removed from the rescued receptor. I have the
• the methods disclosed herein can be used to compare a subject before and after treatment. For example, binding of a GnRHR agonist to a mutant GnRHR, GnRH agonist-stimulated IP production, and the surface- bound mutant GnRHR-ligand complex internalized can be determined using the methods described in Examples 1 and 2.
• Translocation The transport of an agent, such as a wild-type or mutant GnRHR, from one part of a cell to another part of the cell. In one example, it includes the transport of a wild-type or mutant GnRHR across a cell membrane.
• the mutant GnRHR is a human GnRHR including a N 10 K, T 32 I, E 90 K, Q 106 R, A 129 D, R 139 H, C 200 Y, R 262 Q,
• the mutant GnRHR is a rat GnRHR including a Des325-327, DesL237-L241 or a C278A mutation.
• the agents to be screened are GnRHR antagonists.
• Exemplary antagonists include, but are not limited to derivatives, analogs, and mimetics of indole, quinolone, and macrolide antibiotics, such as Q89, (7-chloro-2-oxo-4- ⁇ 2-[(2S)- piperidin-2-yl]ethoxy ⁇ -N-pyrimidin-4-yl-3-(3,4,5-trimethylphenyl)- 1 ,2- dihydroquinoline-6-carboxamide); Q76, (N-(7-chloro-3-(3,5-dimethylphenyl)-2-oxo-4- ⁇ 2-[(2S)-piperidin-2-yl]ethoxy ⁇ -l,2-dihydroquinolin-6-yl)-N-cyclopropylurea); Q08, ((2S)-2-(2- ⁇ [7-chloro-6-[(6,7-dimethoxy-3,4-dihydroisoquinol
• indoles and quinolones to be screened have an IC 50 of less than 5 nM for wild-type human GnRHR, for example less than 4 nM, less than 3 nM, less than 2.5 nM, less than 2.3 nM, less than 2 nM, or less than 1 nM.
• the macrolide antibiotic to be screened such as an erythromycin-derived macrolide, has an IC 50 of less than 700 nM for wild-type human GnRHR, for example less than 100 nM, less than 25 nM, less than 20 nM, or less than 5 nM.
• the method includes screening an indole, quinolone, or macrolide (including derivatives, analogs, or mimetics thereof) for an ability to restore functionality to a mutant GnRHR.
• the method includes include contacting the indole, quinolone, or macrolide with the mutant GnRHR under conditions that allow interaction between the indole, quinolone, or macrolide and the mutant GnRHR and under conditions that restore function to the mutant GnRHR, then incubating the indole, quinolone, or macrolide with the mutant GnRHR for a time sufficient to allow interaction between the indole, quinolone, or macrolide and the mutant GnRHR.
• the mutant GnRHR is expressed recombinantly in a cell, and the cell is contacted with the agent. Subsequently, a determination is made as to whether functionality was restored to the mutant GnRHR. Agents that restore functionality to mutant GnRHR can be selected for further study.
• the method includes determining an amount of inositol phosphate (IP) production by the cell, wherein an increase in IP production as compared to an amount of IP production in the absence of the indole, quinolone, or macrolide indicates that the agent restored functionality to the mutant GnRHR.
• IP production increases by at least 25%, such as at least 50%, at least 75%, at least 100%, at least 200%, or even at least 1000% in the presence of the agent, as compared to an amount of IP production in the absence of the agent. Any method used by those skilled in the art can be used to measure IP production.
• IP production is measured as follows. Following incubating the agent (such as an indole, quinolone, or macrolide) with the cell, the cell is washed to remove the agent, then incubated with [ 3 H]inositol (a precursor of IPs) for a time sufficient to allow uptake of the inositol by the cell. The cell is then contacted the cell with a GnRHR agonist for a time sufficient to allow stimulation of IP production, and the amount of IP produced determined, for example by disrupting the cells and the amount of radioactivity incorporated into total IPs is determined by liquid scintillation spectroscopy (for example, see the method described in Huckle and Conn, Methods Enzymol 141:149-55, 1987).
• agent such as an indole, quinolone, or macrolide
• Exemplary GnRHR agonists include buserelin (D-tert-butyl-Ser 6 , des-Gly 10 , Pro 9 , ethylamide-GnRH), leuprolide (D-Leu 6 , Pro 9 , des-Gly 1 °-ethylamide-GnRH), and GnRH.
• Another assay that can be used to determine whether functionality was restored to the mutant GnRHR includes determining an amount of GnRHR agonist binding to a mutant GnRHR on a surface of the cell, wherein an increase in binding as compared to an amount of binding in the absence of the indole, quinolone, or macrolide indicates that the agent restored functionality to the mutant GnRHR.
• GnRHR agonist binding increases by at least 25%, such as at least 50%, at least 75%, at least 100%, at least 200%, or even at least 1000% in the presence of the agent, as compared to an amount of binding in the absence of the agent. Any method used by those skilled in the art can be used to measure GnRHR agonist binding.
• GnRHR agonist binding is measured as follows. Following incubating the agent (such as an indole, quinolone, or macrolide) with the cell, the cell is washed to remove the agent, then incubated with a GnRHR agonist (such as a GnRHR ligand) of the for a time sufficient to allow binding of the agonist to GnRHR present on the cell surface. Subsequently, the amount of GnRHR agonist binding is determined, for example by detecting a label present on the agonist.
• the GnRHR agonist includes a label, such as a radiolabel or fluorophore.
• the GnRHR agonist includes 125 I-buserelin.
• Another assay that can be used to determine whether functionality was restored to the mutant GnRHR includes determining an amount of internalized surface-bound mutant GnRHR-ligand complex, wherein an increase in internalization as compared to an amount of internalization in the absence of the indole, quinolone, or macrolide indicates that the agent restored functionality to the mutant GnRHR.
• internalization of surface-bound mutant GnRHR-ligand complex increases by at least 25%, such as at least 50%, at least 75%, at least 100%, at least 200%, or even at least 1000% in the presence of the agent, as compared to an amount of internalization in the absence of the agent.
• any method used by those skilled in the art can be used to measure internalization of surface-bound mutant GnRHR-ligand complex.
• internalization of surface-bound mutant GnRHR-ligand complex is measured as follows. Following incubating the agent (such as an indole, quinolone, or macrolide) with the cell, the cell is washed to remove the agent, then incubated with a GnRHR agonist (such as buserelin) for a time sufficient to allow binding of the agonist to GnRHR present on the cell surface, thereby forming a GnRHR-agonist complex, and allowing the complex to internalize into the cell.
• the agent such as an indole, quinolone, or macrolide
• the agonist is removed from the cell surface, and the amount of GnRHR-agonist complex present in the cell determined, for example by detecting a label present on the agonist.
• the GnRHR agonist includes 125 I-buserelin, and internalization is determined by quantitating the amount of radioactivity present.
• the method includes contacting the indole, quinolone, or macrolide with a wild-type GnRHR under conditions that allow interaction between the indole, quinolone, or macrolide and the wild-type GnRHR, in the presence or absence of a GnRHR agonist, and then determining an inhibitory concentration (IC 50 ) of the indole, quinolone, or macrolide for the wild-type GnRHR.
• IC 50 inhibitory concentration
• Methods for determining the ICs 0 of an antagonist include determining the specificity of the agent for GnRHR, for example by measuring IP production (as described above) in the presence of a range of GnRHR agonist concentrations (such as about 10 "13 M - 10 "7 M) and in the presence of a single concentration of agent (such as about 1 ⁇ g/ml).
• a range of GnRHR agonist concentrations such as about 10 "13 M - 10 "7 M
• a single concentration of agent such as about 1 ⁇ g/ml.
• indoles and quinolones having an IC 50 of less than 3 nM for wild-type GnRHR, and macrolides with an ICso of less than 700 nM for wild-type GnRHR are selected as candidate agents which can restore function to a mutant GnRHR.
• agents identified that were able to restore function to mutant GnRHR were also able to increase the presence of wild-type human GnRHR on the cell surface. Therefore, the methods described above can also be used to screen for agents that increase trafficking of wild-type GnRHR to the cell surface.
• wild-type hGnRHR is incubated with the agent, and the amount of IP production, GnRHR agonist binding, internalization of wild-type GnRHR-ligand complex, or combinations thereof, determined, and compared to an amount in the absence of the agent.
• Such agents can be used to treat subjects having hypogonadism, for example to alleviate symptoms associated with erectile dysfunction, infertility, decreased libido, decrease in beard and growth of body hair, decrease in size or firmness of the testicles, decrease in muscle mass and increase in body fat, enlarged male breast tissue, hot flashes, mood swings, irritability, depression, fatigue, osteoporosis, delayed puberty, or combinations thereof.
• hypogonadism for example to alleviate symptoms associated with erectile dysfunction, infertility, decreased libido, decrease in beard and growth of body hair, decrease in size or firmness of the testicles, decrease in muscle mass and increase in body fat, enlarged male breast tissue, hot flashes, mood swings, irritability, depression, fatigue, osteoporosis, delayed puberty, or combinations thereof.
• Other mutated receptors such as other G-coupled protein receptors.
• cystic fibrosis cystic fibrosis (CFTR chloride channel), systemic amyloidosis (amyloid fibrils, light chain variable domains), I-cell disease, nephrogenic diabetes insipidus (aquaporin-2, V-2 receptor), cancer (p53 protein), retinitis pigmentosa (rhodopsin, carotenoid receptors), emphysema and alpha 1 antitrypsin deficiency liver disease (alpha 1 antitrypsin), Alzheimer's Disease (amyloid, tau protein), Creutzfeldt- Jakob (amyloid), spongiform encephalopathies (prion glycoprotein (PrP)), sickle cell anemia (hemoglobin), Parkinson's Disease (alpha- synclein, Parkin, ubiquitin C) and
• the mutant receptor is a mutant gonadotropin releasing hormone receptors (GnRHRs).
• the mutant human GnRHR includes a N 10 K, T 32 I, E 90 K, Q 106 R, A 129 D, R 139 H, C 200 Y, R 262 Q, L 266 R, C 279 Y or a Y 284 C amino acid substitution.
• the mutant GnRHR is a rat GnRHR including a Des325-327, DesL237-L241 or a C278A mutation.
• the mutant GnRHR is expressed in a cell but is not transported to the cell surface.
• the method includes contacting the cell with a therapeutically effective amount of an agent that increases transport of the mutant GnRHR to the cell surface.
• an agent that increases transport of the mutant GnRHR to the cell surface.
• the agent binds to the GnRHR at a non-ligand binding site, with high affinity.
• Exemplary agents include GnRHR antagonists, such as indoles, quinolones, macrolides, or combinations thereof.
• GnRHR antagonists that at least partially restore function to mutant GnRHR include several members of the indole, quinolone, and macrolide chemical groups, such as Q89, Q76, Q08, In30, In3, A-64755.0, A-177775.0, and A-222509.
• the agent includes an indole antibiotic, quinolone antibiotic, macrolide antibiotic, or combinations thereof.
• the GnRHR antagonist binds to the receptor at its ligand binding site, thereby interfering with binding of the ligand (gonadotropin- releasing hormone, GnRH), the pharmacological chaperone or "pharmacoperone" is removed from the receptor so that the rescued GnRHR can more effectively bind ligand and couple to its effector protein.
• the pharmacological chaperone or "pharmacoperone” is removed from the receptor so that the rescued GnRHR can more effectively bind ligand and couple to its effector protein.
• such antagonists bind to the GnRHR, but do so reversibly, thereby providing a mechanism for removing the therapeutic antagonist.
• a GnRHR antagonist does not bind to the receptor at its ligand binding site, and therefore does not interfere with binding of the ligand once the receptor reaches the cell surface, removal of the antagonist may be less desirable before activating the receptor with an agonist.
• the disclosed methods of restoring function to a mutant GnRHR include contacting a therapeutically effective amount of an indole, quinolone, macrolide, or combinations thereof (including derivatives, analogs, or mimetics thereof) identified using the screening methods described above with the mutant GnRHR, wherein contacting restores function to the mutant GnRHR.
• Restoring function to the mutant GnRHR can include increasing binding of a GnRHR agonist to a mutant GnRHR by at least 25%, increasing GnRH agonist-stimulated IP production by a mutant GnRHR by at least 25%, increasing surface-bound mutant GnRHR-ligand complex internalized by at least 25%, or combinations thereof.
• the mutant GnRHR is present in a subject, and the method includes administering a therapeutically effective amount of the identified indole, quinolone, or macrolide (or combinations thereof) to the subject.
• the indole, quinolone, or macrolide can be administered in a pharmaceutically acceptable carrier, alone or in the presence of additional therapeutic agents.
• the subject has hypogonadotropic hypogonadism (HH).
• the method includes contacting a therapeutically effective amount of an indole, quinolone, macrolide or combinations thereof (including derivatives, analogs, or mimetics thereof) identified using the screening methods described above with the wild-type human GnRHR (hGnRHR), wherein contacting increases wild-type hGnRHR expression at a cell surface.
• the amount of wild-type hGnRHR on the cell surface increases by at least 10%, for example at least 25%, at least 50%, at least 100%, or even at least 150%, in the presence of the agent, as compared to an amount present in the absence of the agent.
• the wild-type hGnRHR is present in a subject, and the indole, quinolone, macrolide or combinations thereof is administered to the subject in a therapeutically effective amount.
• the subject has hypogondadism, and may have one or more of the following symptoms: erectile dysfunction, infertility, decreased libido, decrease in beard and growth of body hair, decrease in size or firmness of the testicles, decrease in muscle mass and increase in body fat, enlarged male breast tissue, hot flashes, mood swings, irritability, depression, fatigue, osteoporosis, delayed puberty, or combinations thereof.
• EXAMPLE 1 Pharmacological Rescue of GnRNR Mutants This example describes methods used to demonstrate that agents from several different chemical classes can restore functionality to several rat and human GnRH mutant receptors.
• agents from several different chemical classes can restore functionality to several rat and human GnRH mutant receptors.
• similar methods can be used to screen other agents of interest, such as other indoles, quinolones, and erythromycin- derived macrolides.
• similar methods can be used to screen and restore function to other mutant G-protein-coupled receptors.
• the agents were utilized; those of the quinolone class are prefaced by the letter "Q” and those of the indole class by the letters "In” and were produced by Merck and Company (Ashton et al. , Bioorg. Med. Chem. Lett. 11 : 1727-31 , 2001 ; Ashton et al. Bioorg. Med. Chem. Lett. 11:1723-6, 2001; and Ashton et al, Bioorg. Med. Chem. Lett.
• A-7662.0 (Erythromycin A); A-64755.0 (11- deoxy-1 l-[carboxy-phenylethylamino]-6-O-methyl-erythromycin A 1 l,12-(cyclic carbamate)); A-177775.0, (3'- ⁇ -desmethyl-3'- ⁇ -cyclopentyl-l 1-deoxy-l l-[carboxy- (3,4-dichlorophenylethylamino)]-6-0-methyl-erythromycin A 1 l,12-(cyclic carbamate)); A-222509.0, 3',3'-N-desmethyl-3',3'-N-cyclopropylmethyl-l 1-deoxy-l 1- [carboxy-(3-chloro,
• GnRHR mutants were constructed, sequenced and prepared using standard molecular biology techniques (Janovick et al, J. Gin. Endocrinol. Metab., 87:3255-62, 2002).
• Manufactured mutants of the rGnRHR included the shortest rat GnRHR c-terminal truncation mutant that results in receptor loss-of-function, des " -rGnRHR; two deletion mutants (des 237"241 -rGnRHR, and des 260"265 -rGnRHR) and two Cys mutants (C 229 A-GnRHR and C 278 A-GnRHR).
• plasmid DNAs were prepared using a Qiagen Endotree Maxi-prep kit (Qiagen, Valencia, CA). The purity and identity of the amplified plasmid DNAs were further verified by restriction enzyme analysis. WT and mutant hGnRHR were separately transiently expressed in COS-7 cells using standard molecular biology methods (Leanos-Miranda et /., J. Gin. Endo. Metab., l:A 25- , 2002).
• DMEM growth medium
• FCS fetal calf serum
• FCS fetal calf serum
• FCS fetal calf serum
• the cells were transfected with 0.05 ⁇ g DNA per well (for IP production assay) or 0.1 ⁇ g DNA per well (for saturation binding studies) using 2 ⁇ l lipofectamine in 0.25 ml OPTI-MEM containing 1% DMSO (vehicle) or 1 ⁇ g/ml of each indole, quinoline or erythromycin macrolide prepared in vehicle.
• IP inositol phosphates
• IP accumulation was measured by Dowex anion exchange chromatography and liquid scintillation spectroscopy, as previously described (Huckle and Conn, Methods Enzymol 141:149-55, 1987). Briefly, 1 ml aliquots of cell lysates were applied to 0.4 ml columns of Dowex 1-X8 (200-400 mesh, formate form). Free [ 3 H]inositol was eluted with 10 bed volumes of water; labeled IP], IP 2 , and IP 3 with 8 bed volumes each of 0.2, 0.5, and 1.0 M ammonium formate in 0.1 M formic acid, respectively. Radioactivity was determined in the various Dowex fractions by liquid scintillation spectroscopy.
• [ 3 H]PIs are not eluted from Dowex columns, and can be identified by thin layer chromatography.
• the IC 50 for each indole, quinoline and erythromycin macrolide was calculated as follows. Cells expressing wild-type GnRHR are incubated in the presence of 1 ⁇ g/ml of the indole, quinoline or erythromycin macrolide and in the presence of various concentrations of I-buserelin (10 " M - 10 " M), and the amount of IP production measured as described above.
• the IC 50 concentration for the unlabeled indole, quinoline or erythromycin macrolide in a competition experiment is the concentration required to inhibit the radiolabeled ligand ( 125 I-buserelin) to the specific ligand binding site of GnRHR by 50%.
• Top is the top of the curve, Bottom is the bottom of the curve; Y is the amount bound (either as cpm or as % of control); and X is the concentration of unlabeled agent.
• the ICs 0 determined for each agent is shown in the FIGS. 1-3.
• FIGS. 1 indoles
• 2 quinolones
• 3 erythromycin macrolides
• the data shown in FIGS. 1-3 are the means + SEM from triplicate determinations. For each chemical class, the data are presented with the lowest IC 50 value (for the hGnRHR, shown in figures) first. The data indicate that a concentration of 1 ⁇ g/ml is, in most cases, the dose of agent that elicits an optimum response.
• FIGS. 4 indoles
• 5 quinolones
• 6 erythromycin macrolides
• FIGS. 7 and 8 show the unrescued coupling of the receptor in the absence (figure 7) or presence (figure 8) of 10 "7 M buserelin, a GnRHR agonist.
• the member of each drug class with the lowest affinity for the human GnRHR is oriented closest to the viewer.
• EXAMPLE 2 GnRHR Internalization in the Presence of GnRH peptide Antagonist This example describes a method that can be used to determine an amount of GnRHR internalization in the presence of a potential therapeutic agent, such as a GnRHR antagonist. Such a method can be used to screen agents for their ability to restore function to mutant GnRHR.
• Cells are transfected with mutant or wild-type GnRHR as described in Example 1.
• 12 well plates were used and 2 x 10 5 cells were plated per well, thereby allowing each plate to serve as one time point.
• the agent such as a GnRHR antagonist or vehicle alone is incubated as described in Example 1 for the IP assay (Example 2).
• a radioligand acid wash method (Marian et al, Mol Pharmacol. 19:399-405, 1981; Heding et al, Endocrinology 141:299-306, 2000) is used to measure internalization of the mutant or WT human GnRHRs. This method distinguishes internalized and non-internalized receptor.
• cells are washed twice with 0.5 ml DMEM containing 0.1 % BSA, then incubated with [ 125 I]-buserelin (see Example 1). At the desired time, the iodinated ligand is removed and the plate placed on ice. Cells are washed twice with 0.5 ml ice cold PBS, then 0.5 ml acid wash solution (50 mM acetic acid and 150 mM NaCl, pH 2.8) is added to each well and incubated for 12 minutes on ice. To determine the surface-bound iodinated ligand, the acid wash is collected and counted on a Packard gamma counter (Downers Grove, IL).
• a Packard gamma counter (Downers Grove, IL).
• the cells are solubilized in 0.5 ml PBS containing 0.1% Triton-XlOO, collected and counted. Nonspecific binding for all conditions is determined using the same method but in the presence of 10 ⁇ M unlabeled GnRH. Nonspecific binding is subtracted from the surface-bound and internalized radioligand, and the amount of internalized radioligand is expressed as the percent internalized of the total bound at each time point.
• the ability of a mimetic of the indoles, quinolones, and macrolides described in Example 1 to restore function to a GnRHR mutant can be determined using the methods disclosed herein.
• the indole derivative IN3 includes a number of alkyl substituents, specifically methyl groups, such as the gem dimethyl groups and the methyl group at the chiral center adjacent to the indole.
• alkyl substituents may vary in length and position on the molecule, and typically are selected from the group consisting of lower (such as ten carbon atoms or fewer) aliphatic groups, more particularly lower alkyl groups, including straight and branched chains, as well as all biologically active stereoisomers.
• the methyl groups of the 3,5-dimethylbenzene ring can be varied to be other lower aliphatic groups, most likely lower alkyl groups, and the relative positioning of such lower aliphatic groups can be other than 3,5.
• the number of lower aliphatic groups can vary from 1-5.
• IN3 includes an amide functionality, and such amide can vary from the [2.2.2] bicycloaminooctane moiety of IN3.
• other cyclic compounds, as well as acyclic amides may be included.
• the carbonyl oxygen can be replaced with other heteroatoms, most notably sulfur.
• the nitrogen atom of the heterocyclic amines also can vary, and potentially can be replaced with an atom selected from the group consisting of oxygen and sulfur.
• regioisomers of the oxygen, nitrogen and sulfur heterocycles can replace the heterocyclic amine moieties.
• the indole moiety may be replaced by fused bicyclic aromatic moieties, such as benzimidazole, benzofuran, and benzothiophene derivatives.
• aromatic heterocycles are selected from the group consisting of furan, pyrrole, thiophene, oxazole, imidazole, thiazole, quinoline, isoquinoline, pyrimidine, purine, benzofuran, benzothiophene, and derivatives thereof.
• the hydrogen atom bonded to the nitrogen atom of the aliphatic amine also can be replaced with lower aliphatic substituents, with such substituents typically being selected from the group consisting of lower alkyl groups, with methyl groups being a likely such substituent.
• the substitution pattern of the pyridine derivative can be varied, for example a 2- pyridine or a 3 -pyridine derivative can be used in place of the 4-pyridine moiety of IN3. Additionally, the pyridine moiety can be replaced with another aromatic group, such as a five membered, six membered, or fused aromatic heterocycle. Finally, the number of methylene units spacing particular functional groups and moieties of IN3 can be varied. For example, IN3 includes 2 methylene units that space the amine portion of IN3 from the pyridine ring. The number of methylene units may vary from about 1-10, more typically from about 2-5.
• EXAMPLE 4 Screening Assays This example describes methods that can be used to screen agents for their ability to restore functionality to a mutant receptor, such as GnRHR having one or more amino acid substitutions or deletions that lead to the development of HH in a subject.
• a mutant receptor such as GnRHR having one or more amino acid substitutions or deletions that lead to the development of HH in a subject.
• agents from several different chemical families including indoles, quinolones, and macrolides, were able to restore function to many GnRHR mutants. Therefore, screening assays can be used to identify and analyze other agents, such as other GnRHR antagonists, for example other derivatives of indoles, quinolones, and macrolides, that can also restore function to mutant GnRHR.
• the present disclosure is not limited to the particular methods disclosed herein.
• Agents identified via the disclosed assays can be useful, for example, in restoring function to mutant GnRHR molecules, for example in treating a subject ' having HH.
• agents identified via the disclosed assays can be useful, for example, in increasing cell-surface expression of wild-type GnRHR, for example in treating a subject having hypogonadism. Assays for testing the effectiveness of the identified agents, are discussed below.
• agents that can be screened include, but are not limited to, any peptide or non-peptide composition in a purified or non-purified form, such as peptides made of D-and/or L-configuration amino acids (in, for example, the form of random peptide libraries; see Lam et al, Nature 354:82-4, 1991), phosphopeptides (such as in the form of random or partially degenerate, directed phosphopeptide libraries; see, for example, Songyang et al, Cell 72:767-78, 1993), antibodies, and small or large organic or inorganic molecules.
• a test agent can also include a complex mixture or "cocktail" of molecules. Particular test agents include indole, quinolone, and macrolide derivatives and mimetics.
• the basic principle of the assay systems used to identify agents that restore function to mutant GnRHR, increase expression of wild-type GnRHR to the cell surface, or both involves preparing a reaction mixture containing the wild-type or mutant GnRHR protein and the agent under conditions and for a time sufficient to allow the GnRHR and agent to interact and bind and restore function to the mutant GnRHR (or increase expression of the wild-type protein on the cell surface, or both). Controls are incubated without the test agent or with a placebo. Exemplary controls include agents known not to bind to or restore function to GnRHR, such as A-7662.0, Q08, and IN3 lb.
• the ability of the agent to restore function to the mutant GnRHR (or increase expression of the wild-type protein on the cell surface, or both) is then determined.
• the ability of the agent to increase expression of the wild-type protein on the cell surface by at least a desired amount indicates that the agent can be used to treat a subject having hypogonadism, such as secondary hypogonadism.
• a desired amount such as an increase of at least 10%, at least 20%, or even at least 50% in the presence of the agent as compared to an amount of cell-surface expression in the absence of the agent.
• Methods that can be used to assess these GnRHR activities are described in Examples 1 and 2. Briefly, cells expressing a GnRHR protein (wild-type or mutant) are contacted with the agent.
• the amount of agent administered can be determined by skilled practitioners. In some examples, several different doses of the potential therapeutic agent can be administered, to identify optimal dose ranges.
• assays are conducted to determine the amount of binding of a GnRHR agonist to a mutant GnRHR, the amount of GnRH agonist-stimulated IP production by a mutant GnRHR, the amount of surface-bound mutant GnRHR-ligand complex internalized, the amount of wild-type GnRHR on the cell surface, or combinations thereof, using the methods described in Examples 1 and 2.
• an agent such as those identified using the methods provided above, to restore function to a mutant GnRHR, to increase cell-surface expression of wild-type GnRHR, or both, can be assessed in animal models.
• An animal model of hypogonadism, such as HH can be made using standard methods known in the art (for example, see U.S. Patent Nos. 6,037,521; 6,323,390; and 6,576,811, all herein incorporated by reference). Briefly, a gene-targeting vector is generated using standard molecular biology methods. The targeting vector is transfected into ES cells, for example by electroporation. Surviving clones are selected, and analyzed by Southern blot to identify homologous recombinants.
• Homologous recombinants are expanded, re-verified by Southern blot, and used to generate mouse chimeras by blastocyst injection. The resulting chimeras are crossed, and the resulting progeny tested to identify those that are heterozygous for the mutation. Heterozygotes are crossed to generate homozygous mutants.
• the resulting homozygous mutant animal models can also be used to screen agents for an ability to ameliorate symptoms associated with hypogonadism.
• animal models can be used to determine the LDso and the ED 50 in animal subjects, and such data can be used to determine the in vivo efficacy of potential agents.
• Animals of any species including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates, such as baboons, monkeys, and chimpanzees, can be used to generate an animal model of hypogonadism if needed.
• the appropriate animal is inoculated with the agent identified in the examples above, alone or in combination with other therapeutic agents.
• the amount of agent administered can be determined by skilled practitioners. In some examples, several different doses of the potential therapeutic agent can be administered to different test subjects, to identify optimal dose ranges. Subsequent to the treatment, animals are observed for the symptoms associated with hypogonadism. A decrease in the symptoms associated with hypogonadism in the presence of the agent provides evidence that the agent is a therapeutic agent that can be used to restore function to mutant GnRHR or increase expression of wild-type GnRHR, or both.
• This example describes methods that can be used to increase expression of a wild-type hGnRHR on the cell surface, for example to treat, or reduce the symptoms of hypogonadism, such as erectile dysfunction, infertility, decreased sex drive, decrease in beard and growth of body hair, decrease in size or firmness of the testicles, decrease in muscle mass and increase in body fat, enlarged male breast tissue, hot flashes, mood swings, irritability, depression, fatigue, osteoporosis, delayed puberty, or combinations thereof.
• hypogonadism such as erectile dysfunction, infertility, decreased sex drive, decrease in beard and growth of body hair, decrease in size or firmness of the testicles, decrease in muscle mass and increase in body fat, enlarged male breast tissue, hot flashes, mood swings, irritability, depression, fatigue, osteoporosis, delayed puberty, or combinations thereof.
• hypogonadism such as erectile dysfunction, infertility,
• agents as well as other indoles, quinolones or macrolides, such as agents identified using the methods described in Example 4 can be administered to a subject at a therapeutically effective dose, thereby relieving the symptoms associated with hypogonadism (for example as sometimes found in older men and women).
• agents can also be administered with other therapeutic agents, such as testosterone and estrogen.
• the agent administered is a GnRHR antagonist
• the antagonist is removed from the GnRHR at the cell surface, to facilitate binding of the receptor to its ligand, GnRH.
• Methods for removing GnRHR at the cell surface include using an antagonist that reversibly binds to the receptor, and allowing the antagonist to fall off of the receptor over time.
• HH Hypogonadotropic Hypogonadism
• the agent administered is a GnRHR antagonist
• the antagonist is removed from the GnRHR at the cell surface as described in Example 5.
• EXAMPLE 7 Calculation of IC 50 This example describes a method that can be used to determine the inhibitory concentration (IC 5 o) of an agent, such as a GnRHR antagonist. However, one skilled in the art will appreciate that other methods can be used.
• Agents can be tested over a range of concentrations (for example, 10 "1 to 10 "8 ⁇ M).
• concentrations for example, 10 "1 to 10 "8 ⁇ M.
• serial 10-fold dilutions of each agent are prepared in a vehicle (such as DMSO) and stored on ice.
• Cells such as COS-7 cells, are transfected with wild-type or mutant hGnRHR as described in Example 1. Cells are transfected with about 0.01 ⁇ g - 0.05 mg DNA for inositol phosphate production, or 0.1 mg DNA for saturation binding studies.
• Cells expressing wild-type GnRHR are incubated in the presence of the agent, such as 1 ⁇ g/ml, and in the presence of various concentrations of 125 I- buserelin (10 " M - 10 " M), and IP production or saturation binding measured as described in Example 1.
• the most accurate method for determining IC 0 values is to use non-linear regression analysis, as described in Example 1.
• compositions and Modes of Administration This example provides methods and pharmaceutical compositions that can be used to administer a pharmacological chaperone (pharmacoperone) (alone or in combination with other therapeutic agents) that can restore function to mutant GnRHR, to increase expression of wild-type GnRHR at the cell surface, or both.
• pharmacoperone is a GnRHR antagonist.
• Administration of such compositions to a subject can begin whenever treatment of symptoms associated with decreased binding of GnRH to its receptor, for example due to expression of a mutant GnRHR, is desired.
• compositions that include a pharmacoperone may typically be used to treat human subjects, they can also be used to treat similar or identical diseases in other vertebrates such as other primates, farm animals such as swine, cattle and poultry, and sport animals and pets such as horses, dogs and cats.
• compositions that include a pharmacoperone can be formulated in unit dosage form, suitable for individual administration of precise dosages.
• a therapeutically effective amount of a pharmacoperone can be administered in a single dose, or in multiple doses, for example daily, during a course of treatment.
• Compositions that include a pharmacoperone can be administered whenever the effect (such as decreased symptoms of HH) is desired.
• a time-release formulation can also be utilized.
• a therapeutically effective amount of a composition that includes a pharmacoperone can be administered as a single pulse dose, as a bolus dose, or as pulse doses administered over time.
• pulse doses a bolus administration of a composition that includes a pharmacoperone is provided, followed by a time-period wherein no pharmacoperone is administered to the subject, followed by a second bolus administration.
• pulse doses of compositions that include a pharmacoperone are administered during the course of a day, during the course of a week, or during the course of a month.
• compositions including a pharmacoperone can depend on the molecule utilized, the subject being treated, the severity and type of the affliction, and the manner of administration, and should be decided according to the judgment of the practitioner and each subject's circumstances.
• Therapeutically effective amounts of compositions that include a pharmacoperone are those that restore function to a mutant GnRHR by a desired level, or that increase expression of wild-type GnRHR on the cell surface, or both.
• In vitro assays can be employed to identify optimal dosage ranges. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
• a therapeutically effective amount of a pharmacoperone can vary from about 0.001 ⁇ g per kilogram (kg) body weight to about 20 mg per kg body weight, such as about 1 ⁇ g to about 5 mg per kg body weight, such as about 2 ⁇ g to about 0.5 mg per kg body weight, or about 5 ⁇ g to about 1 mg per kg body weight.
• the exact dose is readily determined by one of skill in the art based on the potency of the specific compound (such as fludrocortisone or a mimetic thereof) utilized, the age, weight, sex and physiological condition of the subject.
• compositions or pharmaceutical compositions can be administered by any route, including intravenous, intraperitoneal, subcutaneous, sublingual, transdermal, intramuscular, oral, topical, transmucosal, vaginal, nasal, rectal, by pulmonary inhalation, or combinations thereof.
• Compositions useful in the disclosure may conveniently be provided in the form of formulations suitable for parenteral (including intravenous, intramuscular and subcutaneous), nasal, topical, or oral administration.
• parenteral refers to non-oral modes of administration that include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
• compositions that include a pharmacoperone are administered in combination with (such as before, during, or following) a therapeutically effective amount of one or more other therapeutic agents, such as a steroid (for example estrogens and androgens) or other agents that alleviate symptoms associated with hypogonadism, in a single composition or solution for administration together.
• a steroid for example estrogens and androgens
• Compositions that include a pharmacoperone can be administered simultaneously with the additional agent(s), or administered sequentially.
• a composition that includes a pharmacoperone is formulated and administered with estrogen or androgen as a single dose.
• compositions can be provided as parenteral compositions, such as for injection or infusion.
• Such compositions are formulated generally by mixing a pharmacoperone at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, for example one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
• a pharmacoperone can be suspended in an aqueous carrier, for example, in an isotonic buffer solution at a pH of about 3.0 to about 8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0.
• Useful buffers include sodium citrate-citric acid and sodium phosphate-phosphoric acid, and sodium acetate/acetic acid buffers.
• the active ingredient optionally together with excipients, can also be in the form of a lyophilisate and can be made into a solution prior to parenteral administration by the addition of suitable solvents. Solutions such as those that are used, for example, for parenteral administration can also be used as infusion solutions.
• a form of repository or "depot” slow release preparation can be used so that therapeutically effective amounts of the preparation are delivered into the bloodstream over many hours or days following transdermal injection or delivery.
• Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
• the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
• Pharmacoperones can be utilized as free bases, as acid addition salts or as metal salts.
• the salts ideally are pharmaceutically acceptable, and include metal salts, for example alkali and alkaline earth metal salts, such as potassium or sodium salts. Numerous pharmaceutically acceptable acid addition salts are available. Such products are readily prepared by procedures well known to those skilled in the art.
• compositions that include a pharmacoperone as an active ingredient can be formulated with an appropriate solid or liquid carrier, depending upon the particular mode of administration chosen.
• the product can be shaped into the desired formulation.
• the carrier is a parenteral carrier, such as a solution that is isotonic with the blood of the recipient.
• carrier vehicles include water, saline, Ringer's solution, glycerol and dextrose solution.
• Non- aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.
• Other carriers include, but are not limited to: fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, also binders, such as starches, for example corn, wheat, rice or potato starch, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/or polyvinylpyffolidone, and/or, if desired, disintegrators, such as the above-mentioned starches, also carboxymethyl starch, cross-linked polyvinylpyrrolidone, alginic acid or a salt thereof, such as sodium alginate.
• fillers such as sugars, for example lactose, saccharose, mannitol or sorbitol
• cellulose preparations and/or calcium phosphates for example tricalcium phosphate or calcium hydrogen phosphate
• binders such as starches, for
• the disclosed pharmaceutical compositions can also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
• auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
• Excipients that can be included in the disclosed compositions include flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol, or derivatives thereof.
• compositions including a pharmacoperone can be administered by sustained- release systems.
• sustained-release systems include suitable polymeric materials (such as, semi-permeable polymer matrices in the form of shaped articles, for example films, or mirocapsules), suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt).
• Sustained-release compositions can be administered orally, parenterally, intracistemally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), or as an oral, otic, or nasal spray.
• Sustained-release matrices include polylactides (U.S. Patent No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556, 1983, poly(2-hydroxyethyl methacrylate)); (Langer et al, J. Biomed. Mater. i?as.15: 167-277, 1981; Langer, Chem. Tech. 12:98- 105, 1982, ethylene vinyl acetate (Langer et al, Id.) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988).
• polylactides U.S. Patent No. 3,773,919, EP 58,481
• copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al.,
• Sustained-release compositions include liposomes containing a pharmacoperone (see generally, Langer, Science 249:1527-1533, 1990; Treat et al, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317-327 and 353-65, 1989).
• Liposomes containing a pharmacoperone thereof can be prepared by known methods: DE 3,218,121; Epstein et al, Proc. Natl Acad. Sci. U.S.A. 82:3688-92, 1985; Hwang et al, Proc. Natl. Acad. Sci. U.S.A.
• compositions for administration can be suitably formulated to give controlled release of a pharmacoperone.
• the pharmaceutical compositions can be in the form of particles comprising a biodegradable polymer and/or a polysaccharide jellifying and/or bioadhesive polymer, an amphiphilic polymer, an agent modifying the interface properties of the particles and a pharmacologically active substance. These compositions exhibit certain biocompatibility features that allow a controlled release of the active substance. See U.S. Patent No. 5,700,486.
• compositions that include a pharmacoperone can be delivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201, 1987; Buchwald et al, Surgery 88:507, 1980; Saudek et al., N. Engl. J. Med. 321:5 '4, 1989) or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution can also be employed. One factor in selecting an appropriate dose is the result obtained, as measured by the methods disclosed here, as are deemed appropriate by the practitioner. Other controlled release systems are discussed in Langer (Science 249:1527-33, 1990).
• the pump is implanted (for example see U.S. Patent Nos. 6,436,091; 5,939,380; and 5,993,414).
• Implantable drug infusion devices are used to provide patients with a constant and long-term dosage or infusion of a drug or any other therapeutic agent. Such device can be categorized as either active or passive.
• Active drug or programmable infusion devices feature a pump or a metering system to deliver the agent into the patient's system.
• An example of such an active infusion device currently available is the Medtronic SynchroMedTM programmable pump.
• Passive infusion devices in contrast, do not feature a pump, but rather rely upon a pressurized drug reservoir to deliver the agent of interest.
• An example of such a device includes the Medtronic IsoMedTM.
• the pharmaceutical compositions can take the form of, for example, powders, pills, tablets, or capsules, prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (such as pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (such as lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (such as magnesium stearate, talc or silica); disintegrants (such as potato starch or sodium starch glycolate); or wetting agents (such as sodium lauryl sulphate).
• binding agents such as pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
• fillers such as lactose, microcrystalline cellulose or calcium hydrogen phosphate
• lubricants such as magnesium stearate, talc or silica
• disintegrants such as potato starch or sodium starch glycolate
• wetting agents such as sodium lauryl sulphate
• the compounds for use according to the present disclosure can be conveniently delivered in the fo ⁇ n of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• the dosage unit can be determined by
• the composition of the present disclosure can also be administered as an aerosol or a dispersion in a carrier.
• a pharmacoperone (alone or in combination with other therapeutic agents or pharmaceutically acceptable carriers), is administered as an aerosol from a conventional valve, such as, but not limited to, a metered dose valve, through an aerosol adapter also known as an actuator.
• a suitable fluid carrier can be also included in the formulation, such as, but not limited to, air, a hydrocarbon, such as n-butane, propane, isopentane, amongst others, or a propellant, such as, but not limited to a fluorocarbon.
• a stabilizer is also included, and/or porous particles for deep lung delivery are included (for example, see U.S. Patent No. 6,447,743).
• the method includes administering to a subject having hypogonadism a therapeutically effective amount of a pharmacoperone identified using the methods disclosed herein, such as an indole, quinolone, or macrolide.
• Pharmacoperones can be administered in a single or divided dose. Suitable single or divided doses include, but are not limited to about 0.01, 0.1, 0.5, 1, 3, 5, 10, 15, 30, or 50 ⁇ g/kg/day.
• the disclosure also provides a pharmaceutical pack or kit including one or more containers filled with one or more of the ingredients of the pharmaceutical compositions.
• Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
• Instructions for use of the composition can also be included.
• the disclosure provides compositions that include pharmacoperones, for example a composition that includes at least 50%, for example at least 90%, of a pharmacoperone in the composition. Such compositions are useful as therapeutic agents when constituted as pharmaceutical compositions with the appropriate carriers or diluents.

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• 2004
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