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  • 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
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  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
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Definitions

• This invention relates to the design, development, composition and use of novel calcimimetic molecules able to act in a manner analogous to extracellular calcium ions on cells, to calcilytic molecules which block the activity of extracellular calcium ions on cells, and to methods for their use and identification.
• One such spe ⁇ cialized cell is the parathyroid cell which secretes para ⁇ thyroid hormone (PTH) .
• PTH is the principal endocrine factor regulating Ca 2+ homeostasis in the blood and extracellular fluids.
• PTH by acting on bone and kidney cells, increases the level of Ca 2+ in the blood. This increase in [Ca 2+ ] then acts as a negative feedback signal, depressing PTH secretion.
• Extracellular Ca 2+ acts directly on the parathyroid cell to regulate PTH secretion.
• the existence of a para ⁇ thyroid cell surface protein which detects changes in [Ca 2+ ] has been suggested. This protein acts as a receptor for extracellular Ca 2+ ("the Ca 2+ receptor"), and is sug ⁇ gested to detect changes in [Ca 2+ ] and to initiate a func ⁇ tional cellular response, PTH secretion.
• Measurement of [Ca 2+ ] provides an assay to assess the ability of molecules to act as agonists or antagonists at the Ca 2+ receptor.
• increases in the concentration of extracellular Ca 2+ evoke rapid and transient increases in [Ca 2+ ]j which are followed by lower yet sustained increases in [Ca 2+ ] j .
• the transient increases in [Ca 2+ ]j arise from the mobilization of intracellular Ca 2+ , whereas the lower, sustained increases result from the influx of extracellular Ca 2+ .
• IP 3 inositol-1,4,5- trisphosphate
• diacylglycerol two biochemical indicators which are associated with receptor-dependent mobilization of intracellular Ca 2+ in various other cells.
• various other di- and trivalent cations such as Mg 2+ , Sr 2+ , Ba 2+ , La 3+ , and Gd 3+ also cause the mobilization of intracellular Ca 2+ in parathyroid cells.
• Mg 2 * and La 3+ also increase the formation of IP 3 ; all these inorganic cations depress the secretion of PTH.
• the postulated Ca 2+ receptor on the parathyroid cell is therefore promiscuous because it detects a variety of extracellular di- and trivalent cations.
• SUBSTITUTE SHEET Polyvalent cations [e.g. , divalent and trivalent cations] exert a variety of effects on parathyroid function, such as inhibition of parathyroid hormone (PTH) secretion and cAMP accumulation, stimula ⁇ tion of the accumulation of inositol phos ⁇ phates, and elevation of the cytosolic calcium concentration. These actions are thought to be mediated through a "receptor- like" mechanism. The inhibition of agonist-stimulated cAMP accumulation by divalent and trivalent cations, for exam ⁇ ple, is blocked following preincubation with pertussis toxin.
• the putative polyvalent cation receptor may be coupled to inhibition of adenylate cyclase by the inhibitory guanine nucleotide regulatory (G) protein, G;.
• the Ca 2+ receptor has been presumed to be analogous to other G protein-coupled receptors [e.g., a glycoprotein] , but recent studies with other cell types have raised the possibility that polycations can modulate cell function by alternative or additional mechanisms.
• G protein-coupled receptors e.g., a glycoprotein
• mast cells for example, a variety of amphipathic cations, including mastoparan, a peptide from wasp venom, 48/80, a synthetic polycation, and polylysine, enhance secretion by a pertus ⁇ sis toxin-sensitive mechanism, suggesting the involvement of a G protein.
• No classic cell surface receptor has been identified that could mediate the actions of these diverse agents.
• Polycations have also been shown to interact strongly with acidic phospho- lipids.
• Polylysines of varying chain lengths (20-1000 amino acids) bind to artificial phospholipid vesicles with dissociation constants in the range of 0.5 nM to 1.5 ⁇ M.
• the binding affinity is directly related to the length of the polylysine chain, with polymers of 1000 amino acids having a K ⁇ , of 0.5 nM, shorter polymers having higher Kd values, and lysine not interacting to a significant extent.
• This relationship between potency and chain length is similar to that observed for the effects of polylysine 10200 , polylysine 3g00 , and lysine on parathyroid function.
• RNA isolated from such cells can be expressed in oocytes and caused to provide those oocytes with a phenotype which might be explained by the presence of a Ca 2+ receptor protein.
• Ca 2+ receptor proteins enable certain specialized cells involved in bodily Ca 2+ metabolism to detect and respond to changes in the concen- tration of extracellular Ca 2+ . Although these receptors share certain general characteristics, they can be selec ⁇ tively affected by different pharmacological agents. As detailed below, certain molecules are identified with selective activity on Ca 2+ receptors at parathyroid cells, osteoclasts, and C-cells.
• Ca 2+ receptors constitute discrete molecular targets for a new class of molecules that mimic (“calcimimetics") or antagonize (“calcilytics") the actions of extracellular Ca 2+ .
• Such receptors are present on cell surfaces and have a low affinity for extracellular Ca 2+ (apparent K,, generally greater than about 0.5 mM) .
• Such receptors may include a free or bound effector mechanism, as defined by Cooper, Bloom and Roth, "The Biochemical Basis of Neuropharmacol- ogy", Ch. 4.
• Such receptors are thus distinct from intra- cellular Ca 2+ receptors, e.g.. calmodulin and the tropo- nins.
• Calcimimetics act on Ca 2+ receptors selectively to directly or indirectly depress the function of parathyroid cells or osteoclasts or to stimulate the function of C-cells.
• Calcimimetics and calcilytics of this invention allow novel therapies for hyperparathyroid- ism, osteoporosis and other Ca 2+ -related diseases.
• This application concerns targeting Ca 2+ receptors on each of these three cell types and other cell types that detect and respond to changes in [Ca 2+ ].
• Applicant is the first to demonstrate a Ca 2+ receptor protein in parathyroid cells, and to pharmacologically differentiate such Ca 2+ receptors in other cells, such as C-cells and osteoclasts.
• Applicant is also the first to describe methods by which molecules active at these Ca 2+ receptors can be identified and used as lead molecules in the discovery, development, design, modification and/or construction of useful calcimimetics or calcilytics which are active at Ca 2+ receptors.
• Such calcimimetics or cal ⁇ cilytics are useful in the treatment of various disease states characterized by abnormal levels of one or more components, e.g.. polypeptides such as hormones, enzymes or growth factors, the expression and/or secretion of which is regulated or affected by activity at one or more Ca + receptors.
• the identification of different Ca 2+ receptors in different cell types, and the specific response of such receptors to different lead molecules allows design and construction of specific molecules active in treatment of specific diseases which can be affected by action at such specific Ca + receptors.
• abnormal levels of parathyroid hormone secretion can be affected by such specific molecules without affect ⁇ ing the level of secretion of other Ca 2+ regulated hormones and the like.
• the human parathyroid cell Ca 2+ receptor cDNA can be cloned by screening for functional expression in Xenopus oocytes, and the structural features of organic molecules necessary for activity on the Ca 2+ receptor can be determined through the testing of selected natural products or other molecule libraries and subse ⁇ quent structure-activity studies.
• the invention features a pharmaceutical composition including a molecule which either mimics the activity of extracellular Ca 2+ by evoking an increase in [Ca 2+ ]; in a cell, or blocks an increase in [Ca 2+ ] j elicited by extracellular Ca 2+ .
• the molecule has an EC JO of less than or equal to 5 ⁇ M, and is not protamine.
• mimic is meant that the molecule has one or more of the specific actions of extracellular Ca 2+ on an extra ⁇ cellular Ca 2+ responsive cell. The term does not require that all of the biological functions of extracellular Ca 2+ are mimicked, but rather than at least one such function is mimicked.
• the molecule does not require that the molecule bind to the same site on the receptor as does extracellular Ca 2+ receptor (see for example, the novel compound NPS 467 and its action in Example 20 below) .
• block is meant that one such action of Ca 2+ is reduced or prevented by the molecule.
• the EC J Q can be determined in assays as described below, where the activity mimicked is measured and the concentration of molecule which mimics at half the maximum mimicking effect is the EC ⁇ .
• the IC 50 of a calcilytic is that amount which blocks half maximal activity.
• such assays measure [Ca 2+ ]j increases and are confirmed to be specific to a Ca 2+ recep ⁇ tor by methods described below, or their equivalent.
• bioassays described herein demonstrated that the increase in [Ca 2+ ]i in a cell is transient, having a duration of less than one minute, and the increase in [Ca 2+ ]i is rapid, occurring within thirty seconds; and the molecule also (a) evokes a sustained increase (greater than thirty seconds) in [Ca 2+ ]i, (b) evokes an increase in inositol-l,4,5-trisphosphate and/or diacylglycerol levels, e.g.. within less than 60 seconds, and (c) inhibits dopamine- or isoproterenol- stimulated cyclic AMP formation.
• the tran ⁇ sient increase in [Ca 2+ ] is abolished by pretreatment of the cell for ten minutes with 10 mM sodium fluoride, or the transient increase is diminished by brief pretreatment (not more than ten minutes) of the cell with an activator of protein kinase C, e.g.. phorbol myristate acetate (PMA), mezerein or (-)indolactam V.
• an activator of protein kinase C e.g.. phorbol myristate acetate (PMA), mezerein or (-)indolactam V.
• those molecules which are active in all of the assays described above are particu ⁇ larly useful in this invention since they are specific in their actions to a Ca 2+ receptor of such a cell. This is particularly true for the PMA pretreatment effect described above.
• the cell is a para ⁇ thyroid cell
• the molecule inhibits parathyroid hor- mone secretion from the cell; and the molecule elicits an increase in Cl' conductance in a Xenopus oocyte injected with mRNA from a parathyroid cell, bone osteoclast, juxta ⁇ glomerular kidney cell, proximal tubule kidney cell, kera- tinocyte, parafollicular thyroid cell or placental trophoblast.
• the molecule evokes the mobilization of intracellular Ca 2+ to cause the increase in [Ca 2+ ]j;
• the cell is a C-cell or an osteoclast and the molecule inhibits bone resorption in vivo;
• the cell is an osteoclast and the molecule inhibits bone resorption in vitro; or
• the cell is a C-cell and the molecule stimulates calcitonin secretion in vitro or in vivo; and most preferably the molecule is either a calci- mi etic or calcilytic having an EC ⁇ or IC ⁇ at a Ca 2+ receptor of less than or equal to 5 ⁇ M, and even more preferably less than or equal to 1 ⁇ M, 100 nmolar, 10 nmolar, or 1 nmolar.
• Such lower EC ⁇ 's or IC ⁇ 's are advantageous since they allow lower concentrations, of molecules to be used in vivo or in vitro for therapy or diagnosis.
• the discovery of molecules with such low EC ⁇ 's and IC JQ 'S enables the design and synthesis of similarly potent and efficacious molecules.
• calcimimetic molecule which has one or more activities of extracellular Ca 2+ , and preferably mimics the activity of Ca 2+ at a Ca 2+ receptor.
• a parathyroid cell it is a molecule, which when tested on parathyroid cells, in vitro, possesses one or more, and preferably all of the following characteristics as measured by techniques well known to those in the art:
• the molecule causes a rapid (time to peak ⁇ 5 sec) and transient increase in [Ca 2+ ]j that is refrac ⁇ tory to inhibition by 1 ⁇ M La 3+ or Gd 3+ .
• the increase in [Ca 2+ ]i persists in the absence of extracellular Ca 2+ but is abolished by pretreatment with ionomycin (in the absence of extracellular Ca 2+ ) ; 2.
• the increase in [Ca 2+ ] j elicited by extracellular Ca 2+ is not inhibited by dihydropyridines.
• the transient increase in [Ca 2+ ]; caused by the molecule is abolished by pretreatment for 10 min. with 10 mM sodium fluoride; 4.
• the transient increase in [Ca 2+ ] ; caused by the molecule is diminished by pretreatment with an activator of protein kinase C (PKC) , such as phorbol myristate acetate (PMA), mezerein or (-)-indolactam V.
• PKC protein kinase C
• PMA phorbol myristate acetate
• mezerein mezerein
• (-)-indolactam V The overall effect of the protein kinase C activator is to shift the concentration-response curve of the molecule to the right without affecting the maximal response;
• the molecule causes a rapid ( ⁇ 30 sec.) increase in the formation of inositol-l,4,5-trisphosphate and or diacylglycerol; 6.
• the molecule inhibits dopamine- or isoprotere- nol-stimulated cyclic AMP formation;
• the molecule inhibits PTH secretion; 8.
• Pretreatment with pertussis toxin (100 ng/ml for > 4 hrs. ) blocks the inhibitory affect of the molecule on cyclic AMP formation but does not effect increases in [Ca 2+ ] j , inositol-l,4,5-trisphosphate, or diacylglycerol, nor decreases in PTH secretion;
• the molecule elicits increases in Cl " conductance in Xenopus oocytes injected with poly(A) + - enriched mRNA from bovine or human parathyroid cells but is without effect in Xenopus oocytes injected with water or rat brain or liver mRNA;
• the molecule will elicit a response in Xenopus oocytes injected with the specific cDNA or mRNA encoding the receptor.
• calcilytic molecule which blocks one or more of the activities of extracellular Ca 2+ on an extracellular Ca 2+ -sensing cell, preferably by acting as an antagonist at the Ca 2+ receptor.
• a parathyroid cell it is a molecule which, when tested on parathyroid cells in vitro, pos ⁇ sesses one or more, and preferably all of the following characteristics as measured by techniques well known to those in the art:
• the molecule blocks, either partially or com- pletely, the ability of increased concentrations of extra ⁇ cellular Ca 2+ to: a) increase [Ca 2+ ]i, b) mobilize intracellular Ca 2+ , c) increase the formation of inositol-1,4,5- trisphosphate, d) decrease dopamine- or isoproterenol- stimulated cyclic AMP formation, and e) inhibit PTH secretion;
• the molecule blocks increases in Cl " conductance in Xenopus oocytes injected with poly(A) + -mRNA from bovine or human parathyroid cells elicited by extracellular Ca 2+ or calcimimetic compounds but not in Xenopus oocytes injected with water or rat brain or liver mRNA;
• Xenopus oocytes injected with the specific cDNA or mRNA encoding the Ca 2+ receptor, elicited by extracellular Ca 2+ or a calcimimetic compound.
• the Ca 2+ receptor is able to detect and respond to certain inorganic polycations and polycationic organic molecules.
• the parathyroid cell is unable to distinguish increases in extracellular Ca 2+ concentration from the addition of these organic polycations, presumably because these organic molecules act just like extracellu ⁇ lar Ca 2+ at the Ca 2+ receptor.
• Ihe calcimimetic molecules of this invention are particularly good agonists of the Ca 2+ receptor and may be used as drugs that alter selected cellular functions, e.g.. secretion of PTH from para ⁇ thyroid cells. Unlike Ca 2+ most of these molecules act only at one or more, but not all Ca 2+ receptors, and thus provide an ability to specifically target one Ca 2+ receptor.
• the calcimimetics and calcilytics can be formulated as pharmaceutical compositions which are useful for regu ⁇ lating the level of extracellular free Ca 2+ in a patient and for mimicking the effect of extracellular Ca 2+ on a cell selected from the group described above, by adminis ⁇ tering to the patient such a pharmaceutical composition.
• applicant was unaware of any such molecules acting on the Ca 2+ receptor useful in treatment of diseases caused by irregularity in operation or regula ⁇ tion of a Ca + receptor or diseases in an animal having normal Ca 2+ receptors but which can be treated by activat- ing or deactivating such Ca 2+ receptors.
• the molecule has an EC 50 less than or equal to 5 ⁇ M at one or more but not all cells chosen from the group consisting of parathyroid cells, bone osteoclasts, juxtaglomerular kidney cells, proximal tubule kidney cells, keratinocytes , parafollicu ⁇ lar thyroid cells (C-cells) and placental trophoblasts.
• the molecule is positively charged at physiological pH, and is selected from the group consisting of branched or cyclic poly- amines, positively charged polyamino acids, and arylalkyl- amines, e. ⁇ ..
• the branched polyamine has the formula H 2 N- (CH 2 ) .-(NR j -(CH 2 ) ,) k -NH 2 where k is an integer from 1 to 10, each j is the same or different and is an integer from 2 to 20, and each R i is the same or different and is selected from the group consisting of hydrogen and -(CH 2 ) ,-NH 2 , where j is as defined above, and at least one R ⁇ is not hydrogen.
• the molecule has the formula
• each X independently is selected from the group con- sisting of H, CH 3 , CH 3 0, CH 3 CH 2 0, Br, Cl, F, CF 3 , CHF 2 , CH 2 F, CF 3 0, CH 3 S, OH, CH 2 0H, CONH-, CN, N0 2 , and CH 3 CH 2 ;
• Ar is a hydrophobic entity; each R independently is selected from the group consisting of hydrogen, methyl, ethyl, propyl,
• SUBSTITUTE SHEET isopropyl, butyl, isobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, indenyl, indanyl, dihydroindolyl, thiodihydroindolyl, 2-, 3-, or 4-piperid(in)yl;
• Y is selected from the group consisting of CH, nitrogen and an unsaturated carbon;
• Z is selected from the group consist ⁇ ing of oxygen, nitrogen, sulfur.
• each n is independently between 1 and 4 inclusive, and each m is independently between 0 and 5 inclusive.
• the molecule is either a calcimimetic or calcilytic.
• the hydrophobic entity is selected from the group consisting of phenyl, 2-, 3-, or 4-pyridyl, 1- or 2-naphthyl, 1- or 2-quinolinyl, 2- or 3- indolyl, benzyl, and phenoxy;
• the molecule is an R-diphe- nylpropyl- ⁇ -phenethylamine derivative, and the molecule has the formula:
• each X preferably being independently selected from the group consisting of Cl, F, CF 3 , CH 3 , and CH 3 0.
• novel phenyl- ⁇ -phenethylamine analogs and deriva ⁇ tives are provided having the formula:
• alk is straight or branched chain alkylene of from 1 to 6 carbon atoms;
• R 1 is lower alkyl of from 1 to 3 carbon atoms or lower haloalkyl of from 1 to 3 carbon atoms substituted with from 1 to 7 halogen atoms;
• R g and R j are independently selected carbocyclic aryl or cycloalkyl groups, either monocyclic or bicyclic, having 5- or 6- membered rings optionally substituted with 1 to 5 substi ⁇ tuents independently selected from lower alkyl of 1 to 3 carbon atoms, lower haloalkyl of 1 to 3 carbon atoms sub- stituted with 1 to 7 halogen atoms, lower alkoxy of 1 to 3 carbon atoms, halogen, nitro, amino, alkylamino, amido, lower alkylamido of 1 to 3 carbon atoms, cyano, hydroxy, acyl of 2 to 4 carbon atoms lower hydroxyalkyl of 1 to
• Suitable carbocyclic aryl groups are groups having one or two rings, at least one of which having aromatic character and include carbocyclic aryl groups such as phenyl and bicyclic carbocyclic aryl groups such as naphthyl.
• carbocyclic aryl groups such as phenyl and bicyclic carbocyclic aryl groups such as naphthyl.
• the compounds encompassed therein may exist as racemic mixtures and as individual stereoisomers.
• R-phenylpropyl ⁇ -phenethylamine derivatives which are believed to exhibit enhanced activity in lowering serum ionized calcium.
• Preferred compounds include those where alk is n-propylene. Also preferred are compounds where R t is methyl. Also preferred are those compounds where R 2 and R 3 are optionally substituted phenyl.
• Especially preferred compounds include those where R 2 is monosubstituted phenyl, more preferably meta-substi- tuted.
• Especially preferred R 3 groups include unsubsti- tuted or monosubstituted phenyl, especially ortho-substi- tuted.
• Preferred substitutents for R j include halogen, haloalkyl, preferably trihalomethyl, and alkoxy, prefer ⁇ ably methoxy.
• Preferred substituents for R 3 include halogen.
• the invention features a method for treating a patient having a disease or condi ⁇ tion characterized by an abnormal [Ca 2+ ] or [Ca 2+ ]j in one or more cells or in the blood or plasma or extracellular fluids.
• the method includes the step of administering to the patient a therapeutically effective amount of a mole ⁇ cule which either mimics the activity of extracellular Ca 2+ by evoking an increase in [Ca 2+ ] ; in a cell or blocks an increase in [Ca 2+ ]i elicited by extracellular Ca 2+ .
• abnormal is meant that the patient, compared to the general population, has a different Ca 2+ metabolism that is affected by one or more proteins (e.g.. hormones) in the blood or extracellular body fluids, or other mole ⁇ cules which affect the level of extracellular and/or intracellular Ca 2+ .
• proteins e.g.. hormones
• the diseases include hyperpara- thyroidism, osteoporosis and other bone and mineral- related disorders, and the like (as described, e.g.. in standard medical text books, such as "Harrison's Princi ⁇ ples of Internal Medicine" .
• Such diseases are treated in this invention by molecules which mimic or block one or more of the effects of Ca 2+ and thereby directly or indir ⁇ ectly affect the levels of the proteins or other molecules in the body of the patient.
• therapeutically effective amount is meant an amount that relieves to some extent one or more symptoms of the disease or condition in the patient. Additionally, by “therapeutically effective amount” is meant an amount that returns to normal, either partially or completely, physiological or biochemical parameters associated with or causative of the disease or condition. Generally, it is an amount between about 1 nmole and 1 ⁇ mole of the mole ⁇ cule, dependent on its EC-, and on the age, size, and disease associated with the patient.
• the molecule has an ECg, of less than or equal to 5 ⁇ M, and is not protamine; and most preferably interacts at a Ca + receptor as a calcimimetic or calcilytic. Most preferably the molecule is chosen from one of those described above.
• the patient has a disease characterized by an abnormal level of one or more components the level of which is regulated or affected by activity of one or more Ca 2+ receptors, and the molecule is active on a Ca 2+ receptor of a cell selected from the group consisting of parathyroid cells, bone osteoclasts, juxta ⁇ glomerular kidney cells, proximal tubule kidney cells, keratinocytes, parafollicular thyroid cells, and placental throphoblasts.
• the molecule reduces the level of parathyroid hormone in the serum of the patient, e.g. , to that level present in a normal indi ⁇ vidual, or to a degree sufficient to cause a decrease in plasma Ca 2+ ; and the molecule is provided in an amount sufficient to have a therapeutically relevant effect on the patient.
• the invention features a method for diagnosis of a disease or condition in a patient by identifying the number and/or location (and/or functional integrity) of one or more Ca 2+ receptors within the patient and comparing that number and/or location (and/or func- tional integrity) with that observed in normal patients as an indication of the presence of the disease or condition.
• the method is an immuno- assay in which an antibody to a Ca 2+ receptor is used to identify the number and/or location and/or functional integrity of the Ca 2+ receptors, or the assay involves providing a labelled calcimimetic or calcilytic molecule which binds to a Ca 2+ receptor; and the disease diagnosed is a cancer, e.g., an ectopic tumor of the parathyroid, or a condition characterized by an above normal level in the number of osteoclasts in bone or an increased level of activity of osteoclasts in bone.
• a cancer e.g., an ectopic tumor of the parathyroid, or a condition characterized by an above normal level in the number of osteoclasts in bone or an increased level of activity of osteoclasts in bone.
• the invention features a method for identifying a molecule useful as a therapeutic mole- cule.
• the method includes screening a potentially useful molecule for either an ability to mimic the activity of extracellular Ca 2+ in a cell, or to block an increase in [Ca 2+ ]j elicited by extracellular Ca 2+ , and determining whether the molecule has an ECj o or IC ⁇ of less than or equal to 5 ⁇ M.
• the invention features a recombi ⁇ nant Ca 2+ receptor, a cell including a recombinant Ca 2+ receptor, purified nucleic acid encoding a Ca 2+ receptor, the biological activity and use of the molecule NPS 019, the novel compounds or compositions of matter of NPS 459, NPS 467, NPS 551, and NPS 568 (see Fig. 36) and a method for identifying a useful calcimimetic or calcilytic mole ⁇ cule by identifying a molecule which mimics or blocks one or more activities of Ca 2+ at a first Ca 2+ receptor but not at a second Ca 2+ receptor, e.g.. by use of a recombinant Ca 2+ receptor.
• recombinant is meant to include any Ca 2+ receptor produced by recombinant DNA techniques such that it is distinct from the naturally occurring Ca 2+ receptor either in its location, purity or structure. Generally, such a receptor will be present in a cell in an amount different from those normally observed in nature.
• purified is meant that the antibody or nucleic acid is distinct from naturally occurring antibody or nucleic acid, being separated from antibody or nucleicacid with which it naturally occurs, e.g.. in a vector system, such that it can be used to express recombinant Ca 2+ receptor.
• the antibody or nucleic acid is provided as a homogeneous preparation by standard techniques.
• Such cloned receptors can be expressed in a desired cell, and isolated and crystallized to allow structure determination. Such a structure will allow design of useful molecules of this invention which can bind to the Ca 2+ receptor.
• equivalent such receptors can be cloned using a first clone as a probe for clones in other cell, cDNA or genomic libraries.
• Antibodies to the cloned receptor can be isolated and used as therapeutics in this invention, or as diagnostic tools for determining Ca 2+ receptor numbers and/or loca ⁇ tions and/or functional integrity to diagnose Ca 2+ -related diseases or conditions. Such antibodies can also be used in vivo by intravenous administration as calcimimetics or calcilytics.
• the invention features calcimimetic or calcilytic molecules able to act as either selective agonists or antagonists respectively at a Ca 2+ receptor of one or more but not all cells chosen from the group con ⁇ sisting of parathyroid cells, bone osteoclasts, juxtaglo ⁇ merular kidney cells, proximal tubule kidney cells, kera- tinocytes, parafollicular thyroid cells and placental throphoblasts.
• a composition may include any pharma ⁇ ceutically acceptable carrier known to those in the art to provide a pharmaceutical composition.
• the invention also features modulation of the the number of Ca 2+ receptors in a patient by standard tech- niques, e.g.. antisense and related technologies (e.g.. ribozymes) , as a therapeutic for a disease state.
• This invention provides methods for identifying mole ⁇ cules which affect the activity of a Ca 2+ receptor using assays, as defined below, to detect calcimimetics and/or calcilytics.
• molecules found to be effective to reduce or enhance expression of Ca 2+ receptor at a trans- criptional or translational level by use of the assays or antibodies or other techniques described below can be defined for therapeutic uses.
• FIG. 1 depicts representative molecules useful in the invention.
• Fig. 2 is a graphical representation showing increases in [Ca 2+ ]i induced by extracellular Ca 2+ in quin- 2- or fura-2-loaded bovine parathyroid cells.
• the initial [Ca 2+ ] was 0.5 mM (using CaCl 2 ) and, at each of the arrows, was increased in 0.5 mM increments.
• Fig. 3 is a graphical representation showing mobili ⁇ zation of [Ca 2+ ]j in bovine parathyroid cells.
• the initial [Ca + ] was 0.5 mM and was decreased to ⁇ 1 ⁇ M by the addi- tion of EGTA as indicated.
• Extracellular Mg 2+ (8 mM, final) elicits an increase in [Ca 2+ ]i in the absence of extracellular Ca 2+ .
• Pretreatment with ionomycin (1 ⁇ M) blocks the response to Mg 2+ .
• Pretreatment with 5 ⁇ M molecule 1799 (a mitochondrial uncoupler) is without effect on the response to Mg 2+ .
• Fig. 4 is a graphical representation showing prefer ⁇ ential inhibitory effects of a low concentration of Gd 3+ on steady-state increases in [Ca 2+ ] j and that a high concentra ⁇ tion of Gd 3+ elicits a transient increase in [Ca]j.
• Top panel Control. Initial concentration of extracellular Ca 2+ was 0.5 mM and was increased by 0.5 mM at each of the arrowheads.
• FIG. 5 is a graphical representation showing that the effects of PMA on [Ca 2+ ] j , IP 3 formation, and PTH secretion are overcome by increasing concentrations of extracellular Ca 2+ . For each variable, there is a shift to the right in the concentration-response curve for extracellular Ca 2+ . Note also that the concentration-response curves vary sig- moidally as [Ca 2+ ] increases linearly.
• Fig. 6 is a graphical representation showing that increases in [Ca 2+ ]j elicited by spermine are progressively depressed by increasing [Ca 2+ ].
• Spermine (200 ⁇ M) was added at the time shown by arrowheads. In this and all subsequent figures, the numbers accompanying the traces
• Fig. 7 is a graphical representation showing that spermine mobilizes intracellular Ca 2+ in bovine parathyroid cells.
• EGTA was added to reduce [Ca + ] to ⁇ 1 ⁇ M before the addition of spermine (200 ⁇ M) as indicated (left trace) .
• Figs. 8A and B are graphical representations showing that spermine increases [Ca 2+ ]; and inhibits PTH secretion in bovine parathyroid cells similarly to extracellular
• Fig. 9 is a graphical representation showing the con- trasting effects of PMA on responses to extracellular Ca 2+ and on responses to ATP7S in bovine parathyroid cells.
• SUBSTITUTE SHEET Left panel: The concentration-response curve for extra ⁇ cellular Ca 2+ -induced inhibition of cyclic AMP formation is shifted to the right by PMA (100 nM) . Middle panel: PMA does not affect the ability of ATP ⁇ S to increases [Ca 2+ ]j. Note also that the concentration-response curve to ATP7S shows classical sigmoidal behavior as a function of the log concentration, in contrast to extracellular divalent cations.
• Fig. 10 is a graphical representation showing mobili- zation of intracellular Ca 2+ in human parathyroid cells evoked by extracellular Mg 2+ .
• Cells were obtained from an adenoma and bathed in buffer containing 0.5 mM extracellu ⁇ lar Ca 2+ .
• Fig. 11 is a graphical representation showing mobili ⁇ zation of intracellular Ca 2+ evoked by neomycin or prota- mine in bovine parathyroid cells.
• the ini- tial [Ca 2+ ] and [Mg 2+ ] was 0.5 and 1 mM, respectively.
• the Ca 2+ and Mg + concentrations were increased to 2 and 8 mM, from 0.5 and ImM respectively.
• (c) through (i) neomycin B (30 ⁇ M) or protamine (1 ug/ml) were added as indicated.
• La 3+ (1 ⁇ M) EGTA (1 mM) , or ionomycin (100 nM) were added as indicated.
• Fig. 12 is a graphical representation showing that neomycin B blocks transient but does not block steady- state increases in [Ca 2+ ] £ elicited by extracellular Ca 2+ .
• Bar 1 in.
• Fig. 13 is a graphical representation showing that neomycin B or protamine inhibit PTH secretion at concen ⁇ trations which evoked increases in [Ca 2+ ]j.
• Fig. 14 is a graphical representation showing the preferential inhibitory effects of PMA on cytosolic Ca 2+ transients elicited by spermine.
• Fig. 15 is a graphical representation showing that PMA shifts to the right the concentration-response curves for extracellular Ca 2+ - and neomycin B-induced increases in [Ca 2+ ]j.
• Cells were pretreated with PMA for 1 min. before increasing [Ca 2+ ] or before adding neomycin B as indicated. Each point is the mean + SEM of 3 to 5 experiments.
• Fig. 16 is a graphical representation showing that PMA shifts to the right the concentration-response curves for extracellular Ca 2+ - and spermine-induced inhibition of PTH secretion.
• Cells were incubated with the indicated [Ca 2+ ] and spermine for 30 min. in the presence (closed circles) or absence (open circles) of 100 nM PMA. Each point is the mean + SEM of 3 experiments.
• Fig. 17 is a graphical representation showing that protamine increases the formation of inositol phosphates.
• Parathyroid cells were incubated overnight in culture media containing 4 uCi/ml ⁇ -myo-inositol, washed, and incubated with the indicated concentration of protamine at 37°. After 30 sec. the reaction was terminated by the addition of CHCl 3 :MeOH:HCl and IP X (circles) and IP 3 (triangles) separated by anion exchange chromatography. Each point is the mean of 2 experiments, each performed in triplicate.
• Fig. 18 is a graphical representation showing that PMA depresses the formation of IP] evoked by extracellular Ca 2+ or spermine.
• ⁇ -myo-insoitol-labeled cells were exposed to the indicated [Ca 2+ ] or spermine for 30 sec. before terminating the reaction and determining IP t by anion exchange chromatography. Hatched columns: Cells were pretreated with PMA (100 nM) for 5 min. before increasing [Ca 2+ ] or adding spermine. Each value is the mean of 2 experiments, each performed in triplicate.
• Fig. 19 is a graphical representation showing tran ⁇ sient and sustained increases in [Ca 2+ ]i elicited by neo ⁇ mycin B in human (adenoma) parathyroid cells.
• [Ca 2+ ] was 0.5 mM.
• the sustained increase in [Ca 2+ ]; elicited by neomycin B (10 ⁇ M) was depressed by La 3+ .
• the tran ⁇ sient increase in [Ca 2+ ]i evoked by neomycin B was unaf ⁇ fected by La 3+ .
• Fig. 20 is a graphical representation showing that neomycin B evokes oscillating increases the Cl' conductance in Xenopus oocytes expressing the Ca 2+ receptor.
• Fig. 21 is a graphical representation showing that neomycin B fails to affect basal or evoked increases in C-cells.
• Fig. 22 is a graphical representation showing that extracellular Ca 2+ evokes increases in [Ca 2+ ]j in rat osteo- clasts.
• Fig. 23 is a graphical representation showing that spermine or neomycin B fail to evoke increases in [Ca 2+ ] £ in rat osteoclasts. An indo-1-loaded osteoclast was super ⁇ fused with the indicated concentration of spermine or neomycin B (open bars) alone or together with 20 mM Ca 2+ (solid bars) .
• Fig. 24 is a graphical representation showing the differential effects of argiotoxin (shown as argiopine in the figure, structures also shown in Fig. 1) 659 and argiotoxin 636 on [Ca 2+ ] ; in bovine parathyroid cells.
• the initial [Ca + ] was 0.5 mM and was increased to 1.5 mM where indicated (right trace) .
• argiotoxin 659 300 ⁇ M
• argiotoxin 636 400 ⁇ M
• Fig. 25 is a graphical representation showing that extracellular Mg 2+ or Gd 3+ evoke oscillatory increases in Cl " conductance in Xenopus oocytes injected with bovine parathyroid cell poly(A) + -mRNA.
• trace (a) the concen ⁇ tration of extracellular Ca 2+ was ⁇ 1 ⁇ M and in trace (b) , 0.7 mM.
• Trace (c) shows that extracellular Mg 2+ fails to elicit a response in an oocyte injected only with the mRNA for the substance K receptor, although superfusion with substance K evokes a response. Holding potential was -70 to -80 mV.
• Fig. 26 is a graphical representation showing that extracellular Ca 2+ elicits oscillatory increases in Cl" conductance in Xenopus oocytes injected with human (hyper- plastic) parathyroid tissue poly(A) + -mRNA. The oocyte was tested for responsivity to extracellular Ca 2+ three days
• Fig. 27 is a graphical representation showing the mobilization of intracellular Ca 2+ in bovine parathyroid cells elicited by budmunchiamine.
• Budmunchiamine 300 ⁇ M, structure also shown was added where indicated.
• Fig. 28 is a graphical representation showing that the ability to mobilize intracellular Ca 2+ in parathyroid cells is stereospecific.
• Bovine parathyroid cells loaded with fura-2 were initially suspended in buffer containing 0.5 mM extracellular Ca 2+ before the addition of the indi ⁇ cated concentration of each molecule.
• Fig. 29 is a graphical representation showing effects of La 3+ on [Ca 2+ ] j in osteoclasts. A representative trace from a single rat osteoclast loaded with indo-1 is shown. At low concentrations, La 3+ partially blocks increases in [Ca 2+ ]i, elicited by extracellular Ca 2+ .
• Figs. 30A and B are graphical representations showing the mobilization of intracellular Ca 2+ elicited by extra- cellular Mn 2+ in rat osteoclasts. Extracellular Mn 2+ -evokes concentration-dependent increases in [Ca 2+ ]i (Fig. 30A) that persist in the absence of extracellular Ca 2+ (Fig. 30B) .
• Figs. 31A and 3IB are graphical representations show ⁇ ing mobilization of [Ca 2+ ]j in rat osteoclasts elicited by a molecule termed NPS 449 (see Fig. 38) .
• Isolated rat osteoclasts loaded with indo-1 were superfused with the indicated concentrations of NPS 449 in the presence (Fig. 31A) or absence (Fig. 31B) of 1 mM extracellular CaCl 2 .
• Fig. 32 is a graphical representation showing the mobilization of intracellular Ca 2+ in C-cells evoked by NPS 019 (see Fig. 1) .
• rMTC 6-23 cells were loaded with fura-2 and bathed in buffer containing 0.5 mM [Ca 2+ ] .
• NPS 019 was added to a final concentration of 10 ⁇ M. Representative traces show that the transient increase in [Ca 2+ ]j elicited by NPS 019 is refractory to inhibition by La 3+ (middle trace) and persists in the absence of extracellular Ca + (right trace) .
• Fig. 33 is a graphical representation showing that NPS 456 (Fig. 36) evokes oscillatory increases in Cl" current in Xenopus oocytes which have been injected with bovine parathyroid cell poly(A) + -mRNA.
• Fig. 34 is a graphical representation showing that extracellular Ca 2+ evokes oscillatory increases in Cl " current in Xenopus oocytes which have been injected with human osteoclast mRNA. The oocyte was tested for respon- sivity to extracellular Ca 2+ three days after injection of 50 ng of total poly(A) + mRNA.
• Fig. 35 is a graphical representation showing that the parathyroid cell Ca 2+ receptor is encoded by mRNA in a size range of 2.5-3.5 kb.
• Bovine parathyroid cell poly(A) + -mRNA was size fractionated on denaturing glycerol gra ⁇ terminals and pooled into ten fractions. Each fraction was injected (50 ng/fraction) separately into Xenopus oocytes. After three days, the oocytes were examined for their ability to respond to extracellular Ca 2+ with oscillatory increases in the Cl' conductance.
• Fig. 36 shows the chemical structures of molecules derived from diphenylpropyl- ⁇ -phenethylamine illustrating a family of molecules which were prepared and screened to find the useful molecules of the invention.
• Fig. 37 is a graphical representation showing that
• NPS 021 is a calcilytic compound that blocks the effects of extracellular Ca 2+ on [Ca 2+ ]; in bovine parathyroid cells. Cells were initially bathed in buffer containing 0.5 mM CaCl 2 and, where indicated, the [Ca 2+ ] was increased to a final of 2mM (left trace) . The addition of NPS 021
• Fig. 38 is a graph showing jLn vivo Ca 2+ response to
• SUB S TITUTESHEET Fig. 39 is a graph showing in vivo PTH response to NPS 467.
• Fig. 40 is a graph showing in vivo Ca 2+ response to 25 mg/kg NPS 467.
• Figs. 41 and 42 are graphs showing in vivo Ca 2+ responses to different enantio ers of NPS 467.
• Fig. 43a depicts a reaction scheme for the prepara ⁇ tion of fendiline or fendiline analogs or derivatives depicted in Figure 36.
• Fig. 43b depicts a reaction scheme for the synthesis of NPS 467.
• Fig. 44 depicts a dose response curve showing that NPS 467 lowers serum ionized calcium when administered orally.
• Calcimimetic and Calcilytic Molecules Calcimimetic and calcilytic molecules useful in the invention are generally described above. These molecules can. be readily identified using screening procedures to define molecules which mimic or antagonize the activity of Ca 2+ at Ca 2+ receptors. Examples of such procedures are provided below. These examples are not limiting in the invention but merely illustrate methods which are readily used or adapted by those skilled in the art.
• calcimimetic and calcilytic molecules are identified by screening molecules which are modelled after those described below (called lead molecules) .
• lead molecules As can be seen below there are several specific calcimimetics and calcilytics useful at various Ca 2+ receptors. Derivative molecules are readily designed by standard procedures and tested in one of many protocols known to those skilled in the art. Many molecules may be screened easily to iden ⁇ tify the most useful in this invention.
• Organic cationic molecules which mimic or antagonize the actions of Ca + in other systems contain the requisite structure for activity on a Ca 2+ receptor. Rational design of other useful molecules involves the study of a molecule known to be calcimimetic or calcilytic and then modifying
• poly- amines are potentially calcimimetic since spermine mimics the action of Ca 2+ in several in vitro systems.
• Results show that spermine does indeed cause changes in [Ca 2+ ] £ and PTH secretion pronounced of those elicited by extracellu ⁇ lar di- and trivalent cations (see below) .
• the experi ⁇ ments outlined below are therefore aimed at demonstrating that this phenomenology, obtained with spermine, involves the same mechanisms used by extracellular Ca 2+ .
• bovine parathyroid cells loaded with fura-2 are initially suspended in buffer containing 0.5 mM
• test substance is added to the cuvette in a
• Molecules causing increases in [Ca 2+ ] are subjected to additional testing.
• the two essential characteristics of the molecule important for its consideration as a calci ⁇ mimetic molecule are the mobilization of intracellular Ca 2+ and sensitivity to PKC activators.
• Molecules causing the mobilization of intracellular Ca 2+ in a PMA-sensitive manner have invariably been found to be calcimimetic mole ⁇ cules and to inhibit PTH secretion. Additional testing can, if needed, be performed to solidify this belief. Typically, all the various tests for calcimimetic or calcilytic activity (see above) are not performed.
• a molecule causes the mobilization of intra ⁇ cellular Ca 2+ in a PMA-sensitive manner, it is advanced to screening on human parathyroid cells. For example, mea ⁇ surements of [Ca 2+ ] i are performed to determine the EC ⁇ , and to measure the ability of the molecule to inhibit PTH secretion in human parathyroid cells which have been obtained from patients undergoing surgery for primary or secondary hyperparathyroidism. The lower the EC J Q or IC 50 the more potent the molecule as a calcimimetic or calcilytic.
• SUBSTITUTESHEET a single afternoon, 10-15 molecules can be examined and their ability to mobilize intracellular Ca 2+ (or not) assessed. The sensitivity of any observed increase in [Ca 2+ ]i to depression by PMA can also be assessed. More- over, a single cell preparation can provide data on [Ca 2+ ] £ , cyclic AMP levels, IP 3 and PTH secretion.
• a typical proce ⁇ dure is to load cells with fura-2 and then split the cell suspension in two; most of the cells are used for measure ⁇ ment of [Ca 2+ ]j and the remainder are incubated with mole- cules to assess their effects on cyclic AMP and PTH secre ⁇ tion.
• Parathyroid Cell Preparation Parathyroid glands were obtained from freshly slaugh ⁇ tered calves (12-15 weeks old) at a local abattoir and transported to the laboratory in ice-cold parathyroid cell buffer (PCB) which contains (mM) : NaCl, 126; KCl, 4; MgCl 2 , l; Na-HEPES, 20; pH 7.4; glucose, 5.6, and variable amounts of CaCl 2 e.g.. 1.25 mM.
• PCB parathyroid cell buffer
• Human parathyroid glands, obtained from patients undergoing surgical removal of parathyroid tissue for primary or uremic hyperparathyroid- ism (HPT) were treated similarly to bovine tissue.
• Dissociated parathyroid cells were pre ⁇ pared by collagenase digestion. Dissociated cells were then purified by centrifugation in Percoll buffer. The resultant parathyroid cell preparation was essentially devoid of red blood cells, adipocytes, and capillary tis- sue as assessed by phase contrast microscopy and Sudan black B staining. Dissociated and purified parathyroid cells were present as small clusters containing 5 to 20 cells. Cellular viability, as indexed by exclusion of trypan blue or ethidium bromide, was routinely 95%.
• ITS + is a premixed solution contain ⁇ ing insulin, transferrin, selenium, and bovine serum albu ⁇ min (BSA)-linolenic acid (Collaborative Research, Bedford, MA) .
• BSA bovine serum albu ⁇ min
• the cells were then transferred to plastic flasks (75 or 150 cm 2 ; Falcon) and incubated at 37°C in a humid atmosphere of 5% C0 2 . No serum is added to these overnight cultures, since its presence allows the cells to attach to the plastic, undergo proliferation, and dedifferentiate. Cells cultured under the above conditions were readily removed from the flasks by decanting, and show the same viability as freshly prepared cells.
• Purified parathyroid cells were resuspended in 1.25 mM CaCl 2 -2% BSA-PCB containing 1 ⁇ M fura-2-acetoxymethyles- ter and incubated at 37 ° C for 20 min. The cells were then pelleted, resuspended in the same buffer lacking the ester, and incubated a further 15 min at 37°C. The cells were subsequently washed twice with PCB containing 0.5 mM CaCl 2 and 0.5% BSA and maintained at room temperature (about 20°C) . Immediately before use, the cells were diluted five-fold with prewarmed 0.5 mM CaCl 2 -PCB to obtain a final BSA concentration of 0.1%. The concentration of cells in the cuvette used for fluorescence recording was 1-2 x 10 6 /ml.
• the fluorescence of indicator-loaded cells was mea ⁇ sured at 37°C in a spectrofluorimeter (Biomedical Instru- mentation Group, University of Pennsylvania, Philadelphia, PA) equipped with a thermostated cuvette holder and magne ⁇ tic stirrer using excitation and emission wavelengths of 340 and 510 nm, respectively. This fluorescence indicates the level of cytosolic Ca 2+ . Fluorescence signals were calibrated using digitonin (50 ug/ml, final) to obtain maximum fluorescence (F ⁇ ) , and EGTA (10 mM, pH 8.3, final) to obtain minimal fluorescence (F- ⁇ ,) , and a dissociation constant of 224 nM.
• Leakage of dye is dependent on tem ⁇ perature and most occurs within the first 2 min after warming the cells in the cuvette; dye leakage increases only very slowly thereafter.
• cells were placed in the cuvette and stirred at 37°C for 2-3 min. The cell suspension was then removed, the cells pelleted, and the supernatant returned to a clean cuvette. The supernatant was then treated with digitonin and EGTA as above to obtain as estimate of dye leakage, which is typically 10-15% of the total Ca 2+ - dependent fluorescent signal. This estimate was sub ⁇ tracted from the apparent F ⁇ .
• the amount of PTH in sample super- natants was determined by a homologous radioimmunoassay using GW-1 antibody or its equivalent at a final dilution of 1/45,000.
• 125 I-PTH 65-84; INCSTAR, Stillwater, MN
• Counting of samples and data reduction were performed on a Packard Cobra 5005 gamma counter.
• INS-PTH Nichols Institute, Los Angeles, CA
• cyclic AMP Cells were incubated as above for PTH secretion studies and at the end of incubation, a 0.15 ml sample was taken and transferred to 0.85 ml hot (70°C) water and heated at this temperature for 5-10 min. The tubes were subsequently frozen and thawed several times and the cel- lular debris sedi ented by centrifugation. Portions of the supernatant were acetylated and cyclic AMP concentra ⁇ tions determined by radioimmunoassay.
• Membrane phospholipids were labeled by incubating parathyroid cells with 4 ⁇ Ci/ml ⁇ -myo-inositol for 20-24 hrs. Cells were then washed and resuspended in PCB con ⁇ taining 0.5 mM CaCl 2 and 0.1% BSA. Incubations were per ⁇ formed in microfuge tubes in the absence or presence of various concentrations of organic polycation for different times. Reactions were terminated by the addition of 1 ml chloroform/methanol/12 N HCI (200:100:1; v/v/v) .
• Phytic acid hydrolysate 200 ⁇ l; 25 ⁇ g phosphate/tube water was then added. The tubes were centrifuged and 600 ⁇ l of the aqueous phase was diluted into 10 ml water. Inositol phosphates were separated by ion exchange chromatography using AG1-X8 in either the chloride- or formate-form. When only IP 3 levels were to be determined, the chloride-form was used, whereas the formate form was used to resolve the major inositol phosphates (IP 3 , IP 2 , and IP,) .
• the diluted sample was applied to the chloride-form column and the column washed with 10 ml 30 mM HCI followed by 6 ml 90 mM HCI and the IP 3 eluted with 3 ml 500 mM HCI. The last eluate was diluted and counted.
• the diluted sample was applied to the formate-form column and IP lf IP 2 , and IP 3 eluted sequentially by increasing concentrations of formate buffer. The eluted samples from the formate columns were rotary evaporated, the residues brought up in cocktail, and counted.
• IP 3 The isomeric forms of IP 3 were evaluated by HPLC. The reactions were terminated by the addition of 1 ml 0.45 M perchloric acid and stored on ice for 10 min. Following centrifugation, the supernatant was adjusted to pH 7-8 with NaHC0 3 . The extract was then applied to a Partisil SAX anion-exchange column and eluted with a linear gra- der of ammonium formate. The various fractions were then desalted with Dowex followed by rotary evaporation prior to liquid scintillation counting in a Packard Tri- carb 1500 LSC.
• DMEM Dulbecco's Modified Eagle's medium
• the cells were harvested with 0.02% EDTA/0.05% trypsin, washed twice with PCB containing 1.25 mM CaCl 2 and 0.5% BSA, and loaded with fura-2 as described above for parathyroid cells.
• Rat Osteoclasts were obtained from 1-2 day old Sprague- Dawley rats using aseptic conditions. The rat pups were sacrificed by decapitation, the hind legs removed, and the femora rapidly freed of soft tissue and placed in pre- warmed F-12/DMEM media (DMEM containing 10% fetal calf serumand antibiotics (penicillin-streptomycin-gentamicin; 100 U/ml-100 ug/ml-100 ug/ml)) . The bones from two pups were cut lengthwise and placed in 1 ml culture medium.
• DMEM containing 10% fetal calf serumand antibiotics (penicillin-streptomycin-gentamicin; 100 U/ml-100 ug/ml-100 ug/ml)
• Bone cells were obtained by gentle trituration of the bone fragments with a plastic pipet and diluted with culture medium. The bone fragments were allowed to settle and equal portions (about 1 ml) of the medium transferred to a 6 well culture plate containing 25 mm glass coverslips. The cells were allowed to settle for 1 hr at 37°C in a humidified 5% C0 2 -air atmosphere. The coverslips were then washed 3 times with fresh media to remove nonadherent cells. Measurements of [Ca 2+ ] j in osteoclasts were per ⁇ formed within 6-8 hrs of removing nonadherent cells.
• the cells were superfused with buffer (typically PCB containing 0.1% BSA and 1 mM Ca + ) at l ml/min with or without test sub ⁇ stance.
• buffer typically PCB containing 0.1% BSA and 1 mM Ca +
• the fluorescence emitted by excitation at 340 nm was directed through the video port of the microscope onto a 440 nm dichroic mirror and fluorescence intensity at 495 and 405 nm collected by photomultiplier tubes.
• the out ⁇ puts from the photomultiplier tubes were amplified, digi ⁇ tized, and stored in an 80386 PC. Ratios of fluorescence intensity were used to estimate [Ca 2+ ]i.
• Oocytes were injected with poly(A) + -enriched mRNA from human parathyroid tissue (hyperplastic glands from a case of secondary HPT) . After 3 days, the oocytes were tested for their response to neomycin. Neomycin B evoked oscil ⁇ latory increases in the Cl" conductance which ceased upon superfusion with drug-free saline (see Fig. 20) . Responses to neomycin B were observed at concentrations between 100 ⁇ M and 10 mM. To ensure that the response evoked by neomycin B was contingent upon injection of parathyroid mRNA, the effect of neomycin B on currents in water-injected oocytes was determined.
• neomycin B (10 mM) failed to cause any change in the current. About 40% of oocytes are known to respond to carbachol, an effect mediated by an endogenous muscarinic receptor. In five oocytes examined, one showed inward currents in response to carbachol, and this is shown in the lower trace of Fig. 20. Thus, in cells expressing a muscarinic receptor coupled to increases in [Ca 2+ ] ⁇ and Cl" conductance, neomycin B fails to evoke a response. This shows that the response to neomycin B depends on expression of a specific protein encoded by parathyroid cell mRNA. It suggests quite strongly that in intact cells, neomycin B acts directly on the Ca 2+ receptor to alter parathyroid cell function.
• Certain organic molecules mimic or antagonize the action of extracellular Ca 2+ by acting at the Ca 2+ receptor as shown herein.
• the molecules tested are not necessarily suitable as drug candidates, but they serve to demonstrate that the hypothesis underlying Ca 2+ receptor- based therapies is correct.
• These molecules can be used to determine the structural features that enable them to act on the Ca 2+ receptor, and thus to select molecules useful in this invention.
• An example of one such analytical procedure follows: This example is detailed in the examples below, but is used here to demonstrate the rationale that can be used to design useful molecules of this invention from lead mole ⁇ cules discussed herein. Those in the art will recognize the analytic steps defined in the example and that analo ⁇ gous analysis can be conducted on other lead molecules until the most desired calcimimetic or calcilytic is defined.
• the first series of molecules studied were the amino- glycosides.
• the molecules were examined on bovine para- thyroid cells and their ECjo's for the mobilization of intracellular Ca 2+ determined.
• neomycin B (+6) > gen ⁇ tamicin (+5) bekanamycin (+5) > kanamycin (average +4.5) > streptomycin (+3) > lincomycin (+1) .
• kanamycin which would be predicted to be more potent than strepto ⁇ mycin, is without activity.
• NPS 381 and NPS 382 Two of these molecules, were examined for effects on [Ca 2+ ]j in bovine parathyroid cells.
• the results obtained with these polyamines show that positive charge is not the sole factor contributing to potency.
• argiotoxin 659 contains a 4-hydroxyindole moiety whereas argiotoxin 636 contains a 2,4-dihydroxyphenyl group.
• the net positive charge on these two arylalkylamines is the same (+4) , so their dif- ferent potencies must result from the different aromatic groups. This shows that net positive charge alone does not determine potency.
• the real importance of these find ⁇ ings, however, is the discovery that aromatic groups con- tribute significantly to the ability of molecules to acti ⁇ vate the Ca 2+ receptor.
• Agatoxin 489 (NPS 017) and Agatoxin 505 (NPS 015) both cause the mobilization of intracellular Ca 2+ in para ⁇ thyroid cells with EC ⁇ 's of 6 and 22 ⁇ M, respectively.
• the only difference in the structure of these molecules is a hydroxyl group on the indole moiety (Fig. 1) . This shows that substitutions on the aromatic region of the molecule can influence potency. This indicates that fur ⁇ ther lead molecules to be studied will include those mole- cules having substituted aromatic moieties.
• the structural features to be varied systematically from lead molecules described herein include (1) net posi ⁇ tive charge, (2) number of methylenes separating nitro ⁇ gens, and (3) cyclic versions of, e.g.. polyamines, with and without changes in methylene spacing and net positive charge.
• systematic variations in the struc ⁇ ture and location of aromatic groups can be examined, e.g.. in a variety of arylalkylamines isolated from the venoms of wasps and spiders; and synthetic molecules can be prepared by the coupling of commercially available aromatic moieties to the argiotoxin polyamine moiety.
• the argiotoxin polyamine moiety can be readily coupled to any aromatic moiety containing a carboxylic acid.
• Budmunchiamine A isolated from the plant Albizia amara. is a cyclic derivative of spermine (Fig. 1) .
• the addition of budmunchiamine A to bovine parathyroid cells caused a rapid and transient increase in [Ca 2+ ]i that persisted in the absence of extracellular Ca 2+ and was blunted by pre ⁇ treatment with PMA. It therefore causes the mobilization of intracellular Ca 2+ in parathyroid cells, probably by acting on the Ca 2+ receptor. It is about equipotent with spermine (ECj n about 200 ⁇ M) yet carries one less positive charge (+3) than does spermine.
• budmunchiamine A demon- strate the predictive power of the structure-activity studies and the novel structural information to be gained by testing natural products.
• screening of natural products selected rationally on the basis of the struc ⁇ tural information is readily performed e.g.. molecules can be selected on the basis of well-established chemotaxo- nomic principles using appropriate data bases, such as Napralert.
• macrocyclic polyamine alkaloids derived from papilionoid legumes related to Albizia, such as Pithecolobium and other plant-derived molecules can be screened.
• Fig. 36 provides a second example of a series of molecules which were screened to determine useful mole ⁇ cules of this invention. These molecules were generally derived from fendiline and tested to determine their respective EC 50 's. Moreover, testing of related molecules, such as NPS 447 and NPS 448 reveals stereospecific effects of molecule structure. The most active compounds tested to date are the novel compounds designated NPS 467 and NPS 568 which have EC ⁇ values of less than 5 ⁇ M. Those in the art, by reviewing this series of molecules, can determine other suitable derivatives which can be tested in the invention. These examples demonstrate the general design and screening process useful in this invention, and indicate that additional compound and natural product libraries can be screened as desired by those in the art to determine other useful lead molecules or novel molecules of this invention.
• examples of molecules useful as calcimimetics include branched or cyclic polyamines, posi ⁇ tively charged polya ino acids, and arylalkylamines.
• other positively charged organic molecules including naturally occurring molecules and their analogs, are useful calcimimetics. These naturally occurring mole ⁇ cules and their analogs preferably have positive charge- to-mass ratios that correlate with those ratios for the molecules exemplified herein.
• pre ⁇ ferred naturally occurring molecules and analogs useful as calcimimetics will have a ratio of positive charge: mole ⁇ cular weight (in daltons) from about 1:40 to 1:200, pref ⁇ erably from about 1:40 to 1:100. More specific examples of such molecules are provided below.
• polyamines useful as calcimimetics in this inven ⁇ tion may be either branched or cyclic. Branched or cyclic polyamines potentially have higher calcimimetic activity than their straight-chain analogs. That is, branched or cyclic polyamines tend to have a lower EC ⁇ than their corresponding linear polyamines with the same effective charge at physiological pH (see Table 1) .
• Branched polyamines refers to a chain molecule consisting of short alkyl bridges or alkyl groups joined together by amino linkages, and also con ⁇ taining points at which the chain branches. These "branch points” can be located at either a carbon atom or a nitro ⁇ gen atom, preferably at a nitrogen atom.
• a nitrogen atom branch point is typically a tertiary amine but it may also be quaternary.
• a branched polyamine may have 1 to 20 branch points, preferably 1 to 10 branch points.
• the alkyl bridges and alkyl branches in a branched polyamine are from 1 to 50 carbon atoms in length, preferably from 2 to 6 carbon atoms.
• the alkyl branches may also be interrupted by one or more hetero ⁇ atoms (nitrogen, oxygen or sulfur) or substituted with functional groups such as: halo, including fluoro, chloro, bromo, or iodo; hydroxy; nitro; acyloxy (R'COO-), acylamido (R'CONH-) , or alkoxy (-OR'), where R* may contain from 1 to 4 carbon atoms.
• the alkyl branches may also be substituted with groups that are positively charged at physiological pH, such as amino or guanido. These functional substituents may add or change physical properties such as solubility to increase activity, deliv- ery or bioavailability of the molecules.
• the branched polyamines may have three or more chain and branch termination points. These termination points may be methyl groups or amino groups, preferably amino groups.
• One preferred group of molecules is the group of branched polyamines having the formula:
• each j is the same or different and is an integer from 2 to 20, and each Rj is the same or different and is selected from the group con ⁇ sisting of hydrogen and -(CH 2 ) j -NH 2 , where j is as defined above, and at least one Rj is not hydrogen.
• Particularly preferred branched polyamines of this invention are the molecules N 1 ,N 1 ,N 5 ,N 10 ,N 1 ,N 14 -hexakis-(3- aminopropyl)spermine and N 1 ,N 1 ,N 5 ,N 14 ,N 14 -tetrakis-(3-amino ⁇ propyl)spermine referred to as NPS 381 and NPS 382, respectively, in Figure l.
• Cyclic polyamines refer to hetero- cycles containing two or more heteroatoms (nitrogen, oxy- gen or sulfur) , at least two of which are nitrogen atoms.
• the heterocycles are generally from about 6 to about 20 atoms in circumference, preferably from about 10 to about 18 atoms in circumference.
• the nitrogen heteroatoms are separated by 2 to 10 carbon atoms.
• the heterocycles may also be substituted at the nitrogen sites with aminoalkyl or aminoaryl groups (NH 2 R-) , wherein R is aminoaryl or a lower alkyl of 2 to 6 carbon atoms.
• cyclic polyamines of this invention are shown in Figure 1 as hexacyclen (1,4,7,10, 13,16-hexaaza-cyclooctadecane) and NPS 383.
• Polyamino Acids are shown in Figure 1 as hexacyclen (1,4,7,10, 13,16-hexaaza-cyclooctadecane) and NPS 383.
• the polyamino acids useful in this invention may con ⁇ tain two or more positively charged amino acid residues at physiological pH. These positively charged amino acids include histidine, lysine and arginine. These polypep- tides will vary in length from 2 to 800 amino acids in length, more preferably from 20 to 300 amino acids in length. These polypeptides may consist of a single repeating amino acid residue, or may have the variety of a naturally occurring protein or enzyme.
• the amino acid residues comprising the polyamino acids may be any of the twenty naturally occurring amino acids, or other alternative residues.
• Alternative resi ⁇ dues include, for example, the ⁇ -amino acids of the formula H 2 N(CH 2 ) n COOH, where n is from 2 to 6. These are neutral, nonpolar amino acids, as are sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, norleucine, phenyl glycine, citrulline, methionine sulfoxide, cyclohexyl ala ⁇ nine, and hydroxyproline. Ornithine is an alternative positively charged amino acid residue.
• the polyamino acids of this invention may also be chemically derivatized by known methods.
• Particularly preferred polyamino acids of this inven ⁇ tion include polyarginine, polylysine, and poly(argininyl- tyrosine) , having 20-300 residues.
• Another preferred polyamino acid is protamine, or a protamine analog.
• Arylalkylamines refer to a class of positively-charged toxins derived from arthropod venoms.
• Preferred arylalkylamines of this invention include phil- anthotoxin-433, argiotoxin-636, and argiotoxin-659, aga ⁇ toxin 505, agatoxin 489, the structures of which are shown in Figure 1, and other synthetic molecules modeled after these natural products, such as NPS 019.
• compositions of this invention include not only at least one calcimimetic or calcilytic, but may also include certain additional components.
• additional compo ⁇ nents include targeting components, labels, and other functionalities which may be useful in the applications herein, e.g.. for screening for agonists or antagonists of extracellular Ca 2+ .
• an immunoglobulin or portion thereof, or a ligand specific for parathyroid cells or a Ca 2+ receptor can be used as a target-specific component.
• the immuno ⁇ globulin can be a polyclonal or monoclonal antibody and may comprise whole antibodies or immunologically reactive fragments of these antibodies such as F ⁇ , , F ⁇ , or (F lb ,) 2 .
• Receptor-specific ligands may also be used.
• the compositions of this invention may also contain components derivatized with a molecule or ion which acts as a label.
• a wide variety of labeling moieties can be used, including radioisotopes, chro ophores, and fluores ⁇ cent labels.
• Radioisotope labeling in particular can be readily detected in vivo.
• Radioisotopes may be coupled by coordination as cations in the porphyrin system.
• Useful cations include technetium, gallium, and indium.
• the positively charged molecule can be linked to or associated with a label.
• Chain extensions of 2-4 methylenes were typically accomplished by alkylation with the corresponding N-(bromoalkyl)phthalimide.
• a 1:1.2 mixture of amine to the bromoalkylphthalimide was refluxed in acetonitrile in the presence of 50% KF on Celite.
• Chain extensions were also accomplished by alkylation of a given amine with acrylonitrile or ethylacrylate. Reaction progress was monitored by TLC and intermediates purified on silica gel using combinations of dichloromethane, methanol, and iso- propylamine. Final products were purified by cation exchange (HEMA-SB) and RP-HPLC (Vydac C-18) .
• Amide linkages and cyclizations were typically performed by the reaction of an amine (1° or 2°) with the N-hydroxysuccinimide ester of a given acid. This was accomplished directly (in the case of cyclizations) by treatment of the "amino acid” with dicyclohexylcarbodi- imide under dilute conditions.
• Deprotections of the phthalimido functionality were accomplished by reduction with hydrazine in refluxing methanol.
• Deprotections of the BOC functionality were accomplished in anhydrous TFA.
• Deprotection of benzyl, nitrile, and CBZ protecting functionalities was accomp ⁇ lished by reduction in glacial acetic acid under 55 psi hydrogen in the presence of a catalytic amount of palla- dium hydroxide on carbon.
• Nitrile functionalities in the presence of benzyl and CBZ groups were selectively reduced under hydrogen in the presence of sponge Raney nickel.
• branched polyamines are typically prepared from simple dia inoalkanes of the formula NH 2 -
• the monoprotected product is separated from the unpro ⁇ tected and diprotected products by simple chromatographic or distillation techniques.
• the remaining free amine in the monoprotected product is then selectively alkylated (or acylated) with an alkyl ⁇ ating (or acylating) agent.
• an alkyl ⁇ ating (or acylating) agent to ensure mono-alkylation, the free amine is partially protected by condensation with benzaldehyde followed by sodium borohydride reduction to form the N-benzyl derivative:
• N-benzyl derivative is then reacted with the alkylat ⁇ ing agent.
• a typical alkylating agent is in an N-(bromo- alkyl)phthalimide, which reacts as follows:
• N-(bromobutyl)phthalimide is used to extend or branch the chain with four methylene units.
• reaction with acrylonitrile followed by reduction of the cyano group will extend the chain by three methy ⁇ lenes and an amino group.
• the protecting groups of the resulting chain-extended molecule can then be selectively cleaved to yield a new free amine.
• trifluoroacetic acid is used to remove a BOC group
• catalytic hydrogenation is used to reduce a nitrile functionality and remove a benzyl group
• hydrazine is used to remove phthalimido groups as follows: HydfaZ ⁇ ne
• the new free amine may be aIkylated (or acylated) further as above to increase the length of the polyamine. This process is repeated until the desired chain length and number of branches is obtained. In the final step, deprotection of the product results in the desired poly ⁇ amine. However, further modifications may be effected at the protected end prior to deprotection in the following manner:
• the polyamine is acylated with the N-hydroxysuccinimide ester of 3,4- dimethoxyphenylacetic acid to yield a diprotected polyamine:
• Certain branched polyamines may be formed by simul- taneously alkylating or acylating the free primary and secondary amines in a polyamine formed as above. For example, treatment of spermine with excess acrylonitrile followed by catalytic reduction yields the following: Cyclic polyamines may be prepared as above beginning with starting materials such as hexacylen (Aldrich Chem.) .
• polyamino acids within the scope of the present invention can be made by recombinant techniques known in the art, or may be synthesized using standard solid-phase techniques known in the art. Solid-phase synthesis is commenced from the carboxy-terminal end of the peptide using an ⁇ -amino protected amino acid.
• BOC protective groups can be used for all amino groups even through other protective groups are suitable.
• BOC-lys-OH can be esterified to chloromethylated polystyrene resin supports.
• the polystyrene resin support is preferably a copoly er of styrene with about 0.5 to 2% divinylbenzene as a cross-linking agent which causes the polystyrene polymer to be completely insoluble in certain organic solvents.
• a copoly er of styrene with about 0.5 to 2% divinylbenzene as a cross-linking agent which causes the polystyrene polymer to be completely insoluble in certain organic solvents.
• the polypeptide synthesis may use manual techniques or automatically employing, for example, an Applied Bio- systems 403A Peptide Synthesizer (Foster City, California) or a Biosearch SAM II automatic peptide synthesizer (Bio ⁇ search, Inc., San Rafael, California), following the instructions provided in the instruction manual supplied by the manufacturer.
• an Applied Bio- systems 403A Peptide Synthesizer Fraster City, California
• a Biosearch SAM II automatic peptide synthesizer Bio ⁇ search, Inc., San Rafael, California
• arylalkylamines of the invention are natural products isolated by known techniques, or synthesized as described in Jasys et al.. Tetrahedron Lett. (1988) £9:6223-6226, and Nason et al.. Tetrahedron Lett. (1989) 30:2337-2340.
• One general protocol for preparation of fendiline (or fendiline analogs shown in Fig. 36) is as follows.
• the ether layer was dried with anhydrous K 2 C0 3 and reduced in vacuo.
• the product was then purified by column chromatography or HPLC incorporating silica stationary phase with combinations of CH 2 Cl 2 -Methanol-isopropylamine (typically 3% Methanol and 0.1% isopropylamine in methylene chloride).
• a preferred procedure for preparing fendiline or fendiline analogs uses titanium(IV) isopropoxide and was modified from methods described in J ⁇ . Org. Chem. 5j>:2552 (1990).
• titanium tetrachloride (method described in Tetrahedron Letters .11:5547 (1990)) was used in place of titaniu (IV) isopropoxide.
• the reaction scheme is depicted in Figure 43a.
• R,R' and R" depict hydrocarbyl groups.
• reaction mixture is treated with 1 ml ethanol containing 1 mmole sodium cyano- borohydride (4.) and the resulting mixture is then allowed to stand at room temperature with occasional stirring for about 16 hours. After this time the reaction is quenched by the addition of about 500 ⁇ l water. The reaction mixture is then diluted to about 4 ml total volume with ethyl ether and then centrifuged. The upper organic phase is removed and reduced on a rotovapor.
• the molecules of the inven- tion may be used to: (a) mimic or antagonize one or more of the effect of extracellular Ca 2+ ; (b) affect the extra ⁇ cellular free Ca 2+ level in an individual; and (c) treat diseases such as hyperparathyroidism, osteoporosis and hypertension. While the molecules have generally been shown to have an effect on parathyroid cells, they may also modulate the Ca 2+ receptors on other cells, including bone osteoclasts, juxtaglomerular kidney cells, proximal tubule kidney cells, keratinocytes, parafollicular thyroid cells, and placental trophoblasts.
• the molecules of the invention can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
• the molecules of the invention are formulated in liquid solutions, preferably in physio ⁇ logically compatible buffers such as Hank's solution or Ringer's solution.
• the molecules may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
• Systemic administration can also be by transmucosal or transdermal means, or the molecules can be adminis ⁇ trated orally.
• penetrants appropriate to the barrier to be per ⁇ meated are used in the formulation.
• penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives.
• detergents may be used to facilitate permeation.
• Transmucosal administration may be through nasal sprays, for example, or using suppositories.
• the molecules are formulated into conventional oral administration forms such as capsules, tablets, and tonics.
• the molecules of the invention are formulated into ointments, salves, gels, or creams, as is generally known in the art.
• the amounts of var ⁇ ious compounds of this invention which must be adminis- tered can be determined by standard procedure. Generally it is an amount between about 1 and 50 mg/kg animal to be treated.
• Natural product screening has traditionally provided the lead structures for the development of diverse thera ⁇ Guideic molecules.
• high-throughput screening of natural product libraries or other molecule libraries for activity on the Ca 2+ receptor has not previously been possible.
• the struc- ture of the receptor can additionally be used to gain insight into the molecular geometry of the ligand binding site(s) , and such information used to augment a rational drug design program as discussed above.
• Limited struc ⁇ ture-activity studies and testing of selected natural product molecules will provide the initial structural data base necessary to guide rational natural product screening and drug design.
• the bovine and human parathyroid cell Ca 2+ receptor cDNA can be cloned by functional expression in Xenopus oocytes. It is possible to monitor an increase in intra ⁇ cellular Ca 2+ in Xenopus oocytes indirectly by measuring current through the endogenous Ca 2+ -activated Cl" channel. The amplification of the response afforded by this signal transduction pathway enables the detection of receptor proteins encoded by mRNA at very low levels. This allows the detection of receptor-specific cDNA clones without the need for high affinity ligands, specific antisera, or pro ⁇ tein or nucleic acid sequence information. An example of such a procedure follows.
• Xenopus laevis were obtained from Xeno ⁇ pus I (Ann Arbor, MI) and maintained according to standard procedures.
• Lobes of ovary were excised from hypothermic- ally-anesthetized toads.
• Clusters of oocytes were trans ⁇ ferred into modified Barth's saline (MBS).
• MBS Barth's saline
• Individual oocytes were obtained by incubation in MBS containing 2 mg/ml collagenase (Sigma, Type 1A) for 2h at 21°C and stage V-VI oocytes were selected for injection.
• the capillary tube was then connected to a picospritzer (WPI Instruments) and the volume of the air-pulsed droplets adjusted to deliver 50 ng of mRNA (typically 50 nl) .
• a 35 mm culture dish with a patch of nylon stocking fixed to the bottom was used to secure the oocytes during injection of mRNA into the vegetal pole.
• the injected oocytes were placed into a 35 mm culture dish containing MBS, 100 ug/ml penicillin and 100 U/ml streptomycin and incubated at 18°C for 3 days. Following incubation, an oocyte was placed into a 100 ⁇ l plastic chamber and superfused with MBS at a flow rate of 0.5 ml/min using a peristaltic pump. Test molecules or inorganic polycations were added by rapidly moving the tubing into different buffers. Recording and current- passing electrodes were constructed from thin wall capil ⁇ lary tubing pulled to a resistance of 1-3 Mohms and filled with 3 M KC1.
• Oocytes were impaled (in the animal pole) with both electrodes under microscopic observation and connected to an Axon Instruments Axoclamp 2A voltage-clamp amplifier which was used to set the holding potential (-70 to -80 mV) and to measure the currents that were passed to maintain the holding potential. Currents were recorded directly onto a strip chart recorder.
• tissue was obtained from calves or patients with secondary HPT undergoing surgical removal of the parathyroid glands. Purified cells need not be prepared; whole pieces of gland were used to prepare mRNA that directs the expression of the Ca 2+ receptor in Xenopus oocytes.
• Total cellular RNA was prepared by acid guanidi- nium thiocyanate/phenol extraction of homogenized glands. Oligo-dT cellulose chromatography was used to select poly(A) + mRNA by standard procedures. For size fraction- ation of mRNA, centrifugation through glycerol gradients was used.
• the mRNA was denatured with 20 mM methylmer- curic hydroxide and loaded (50-100 ug at a concentration of l mg/ml) onto a linear 15-30% glycerol gradient pre- pared in Beckman TLS55 tubes. Following centrifugation at 34,000 rpm for 16 hrs, 0.3 ml gradient fractions were col ⁇ lected and diluted in an equal volume of water containing 5mM beta-mercaptoethanol. mRNA was then recovered by two cycles of ethanol precipitation. If desired, the mRNA (50-100 ug of poly(A) + ) may be separated on a 1.2% agarose/ 6.0 M urea preparative gel, along with a range of RNA size markers.
• RNAid binding matrix accordinging to the supplier's standard protocol; Stratagene, Inc.
• Amounts of recovered mRNA were quantified by UV absorbance measurement.
• the size range of mRNA contained within each fraction was determined by formaldehyde/aga ⁇ rose gel electrophoresis using a small quantity (0.5 ug) of each sample.
• the integrity of the mRNA was assessed by in vitro translation of each sample.
• Reticulocyte lysates (commercially available kits; BRL) were used to translate 0.05-0.5 ug of each mRNA fraction.
• the resulting 35 S- labelled proteins were analyzed by SDS-PAGE.
• the intact mRNA was capable of directing the synthesis of proteins of a complete size range, corresponding roughly to the sizes of the individual mRNA fractions.
• a cDNA library was constructed in the vector ⁇ ZAPII, following modifications of the technique of Gubler and Hoffman. RNA from the fraction(s) giving the best response in the oocyte assay was used as starting mater ⁇ ial.
• First-strand cDNA syntheses was primed with an oligo-dT/Notl primer-linker.
• Second-strand synthesis was by the RNase H/DNA Polymerase I self-priming method. Double-stranded cDNA was blunted with T4 DNA polymerase and EcoRI adaptors blunt-end ligated to the cDNA with T4 ligase.
• full-length cDNA was size-selected by exclusion chromatog ⁇ raphy on Sephacryl 500 HA.
• First-strand cDNA was radio- labeled with ⁇ - 32 P-dATP, and all synthesis and recovery steps monitored by following the incorporation of radio ⁇ activity.
• Full-length cDNA recovered from the sizing column was ligated to EcoRI/NotI digested ⁇ ZAPII arms. The ligation mix was test packaged with commercially available high efficiency packaging extract (Stratagene, Inc.) and plated on the appropriate host strain (XL1- blue) .
• the percentage of recombinant phage was determined by the ratio of white to blue plaques when the library is plated on IPTG and X-gal.
• the average insert size was determined from ten randomly selected clones. Phage DNA "mini-preps" were digested with EcoRI and NotI to release the insert, and the size determined by agarose gel electrophoresis.
• the library consisted of >90% recombinant phage, and the insert size ranged from 1.5 to 4.2 kb.
• the recombinant ligation was packaged in large scale to generate 800,000 primary clones. The packaging mix was titered and plated at 50,000 plaques per 15 cm plate. Each pool of 50,000 clones was eluted in SM buffer and stored individually.
• Phage parti ⁇ cles are concentrated by polyethylene glycol precipita ⁇ tion, and phage DNA purified by proteinase K digestion followed by phenol:chloroform extraction. Twenty micro- grams of DNA are digested with NotI. and used as template for in vitro transcription of sense-strand RNA. In vitro transcription is according to standard protocols, utiliz ⁇ ing T7 RNA polymerase and 5' cap analog m 7 GpppG in a 50 ⁇ l total reaction volume. Following Dnase I/Proteinase K digestion and phenol/chloroform extraction, the RNA is concentrated by ethanol precipitation and used for oocyte injection.
• Oocytes were injected with synthetic mRNA (cRNA) from each of the 16 library subpools constituting 50,000 inde ⁇ pendent clones each. After incubation for 3 to 4 days, oocytes were assayed for the ability of 10 mM neomycin to elicit a Ca 2+ dependent Cl-current.
• a pool designated 6 gave a positive signal and thus contains a cDNA clone encoding a functional calcium receptor.
• pool 6 phage were replated at "20,000 plaques per plate and 12 plates harvested. DNA was pre ⁇ pared from each of these subpools and cRNA synthesized.
• oocytes were injected with cRNA and assayed 3-4 days later for the ability of 10 mM neomycin to elicit a Ca 2+ dependent Cl-current.
• a subpool 6-3 was positive and this pool was subjected to a further round of plating reducing the complexity of pools to around 5,000 clones per pool. Pools were again assayed by preparation of cRNA and injection in oocytes.
• a subpool 6-3.4 was positive. In order to expedite further purification of the positive clone in pool 6-3.4, phage DNA from this pool was rescued as plasmid DNA by superinfection with the helper phage, ExAssist.
• Oocytes injected with water did not show any change in the Cl" current when exposed to extracellular Ca 2+ (10 mM) or Mg 2+ (20 or 30 mM) .
• extracellular Ca 2+ (10 mM) or Mg 2+ (20 or 30 mM) were injected with the mRNA encoding the substance K receptor.
• extracellular Mg 2+ (20 mM) did not evoke any current but the cells responded vigor ⁇ ously to the addition of substance K (Fig. 25) .
• results obtained in Xenopus oocytes demonstrate the presence of a mRNA(s) in parathyroid cells encoding a protein(s) which can impart, in normally unresponsive cells, sensitivity to extracellular Ca 2+ .
• the ability of extracellular Mg 2+ to evoke oscillatory increases in Cl' current in the absence of extracellular Ca 2+ demonstrates that the Cl" current depends on the mobilization of intracellular Ca 2+ rather than influx of extracellular Ca 2+ .
• results obtained with La 3+ likewise show that the expressed protein(s) is linked to the mobil ⁇ ization of intracellular Ca 2+ . Together, these data show that the expressed protein(s) acts as a cell surface receptor rather than a channel.
• the mRNA fractions obtained and characterized in the preceding experiments can be assayed by injection into oocytes.
• 10-20 oocytes are injected with 50 ng of RNA at a concentration of 1 ng/nl in water.
• Injected oocytes are maintained at 18° C for 48-72 h after which they are assessed for expression of the Ca 2+ receptor using measurements of Cl" current.
• Cl Cl-dependent Cl
• oocytes are injected with rat liver poly(A) + -enriched mRNA, yeast RNA, or water.
• mRNA in the range of 2.5 - 3.5 kb will encode the receptor.
• mRNA of a larger size may necessitate a cloning approach based on hybrid depletion of parathyroid mRNA prior to oocyte injection. This stra ⁇ tegy is not dependent upon the generation of full-length cDNA clones for success. If receptor expression is not obtained with a single size fraction of mRNA, oocytes are injected with mixed size fractions to determine a combi ⁇ nation that does give rise to a functional receptor. If it does appear that multiple subunits are necessary for the formation of a functional receptor, the hybrid deple ⁇ tion expression cloning strategy is used.
• clones are selected on the basis of their abil- ity to deplete a specific mRNA species from the total mRNA population.
• a clone encoding a single subunit is identi ⁇ fied by its ability to prevent the formation of the active multi-subunit complex.
• exhaustive screening it is pos- sible to identify clones encoding all of the necessary subunits.
• RNA from pools of clones are assayed for their ability to induce expression of the Ca 2+ receptor in Xenopus oocytes by the same techniques used to analyze the original mRNA fractions. Originally, 10 pools representing 100,000 primary clones each are examined. Pools of clones showing a positive response are screened at lower (typically 4 to 10 fold) complexity, and again positive pools further subdivided and screened. This process of library sub-fractionation is followed until individual positive clones are identified.
• anti-sense tran- scripts are generated by T7 transcription of those DNA templates that induce a positive response.
• Anti-sense transcripts are unable to give rise to an authentic recep ⁇ tor, and this will control any non-specific positive sig ⁇ nal arising from injection of synthetic RNA.
• Another concern is the fact that synthetic RNA can occasionally "poison" translation in injected oocytes, by an undefined mechanism.
• synthetic RNAs giving a negative response are co-injected at various dilutions with parathyroid cell mRNA, to determine if they are non-specifically interfering with the expression of the Ca 2+ receptor.
• the cDNA insert When an individual clone encoding the Ca 2+ receptor is identified, the cDNA insert will be excised from the ⁇ vector and used for large scale production of synthetic RNA. Oocyte injection of this single RNA species allows rigorous assessment of the characteristics of the expressed receptor. If the size of the mRNA encoding the Ca 2+ receptor is too large for cloning by direct transcription and expres ⁇ sion, or if multiple subunits are involved, a hybrid- depletion technique of screening pools of clones is used. cDNA insert DNA will be prepared from pools of clones from the size-selected parathyroid cell cDNA library. This DNA is hybridized to parathyroid cell mRNA under conditions that permit the formation of DNA/RNA duplexes.
• the unan- nealed, hybrid-depleted RNA is recovered and used for oocyte injections.
• DNA from pools of clones containing sequences representing Ca 2+ receptor mRNA is depleted from this mRNA from the total parathyroid cell mRNA population, and expression of the receptor is reduced or absent upon oocyte injection.
• a process of sub-fractionation is fol- lowed on pools of clones of decreasing complexity, at each step assaying for cloned DNA that depletes Ca 2+ receptor- encoding mRNA from the total parathyroid cell mRNA popula ⁇ tion.
• the use of an internal control during the hybrid depletion assays ensures that the hybrid-depleted RNA is intact and capable of being translated in the oocyte.
• Human parathyroid cells express a Jeta-adrenergic receptor coupled to adenylate cyclase.
• This receptor can be expressed in oocytes, where it is capable of agonist- induced activation of the endogenous adenylate cyclase.
• oocytes injected with hybrid depleted mRNA are assayed for isoproterenol-induced adenylate cyclase acti ⁇ vation.
• a positive response in this assay serves to indi ⁇ cate that any observed inhibition of Ca 2+ receptor response is specific, and not due to a general inhibition of the total mRNA population.
• the hybrid-depletion screening strategy can result in the isolation of clones that do not contain a complete protein coding region. Positive clones isolated by this screening strategy are sequenced to determine their pro ⁇ tein coding capacity. Northern blot analysis of human parathyroid gland RNA permits the determination of the size of the complete mRNA corresponding to specific clones. If positive clones do not appear to be full- length, the cloned cDNA will be used as a hybridization probe to screen a parathyroid gland cDNA library for complete cDNAs.
• a variety of cell lines are capable of coupling exogenously expressed receptors to endogenous functional responses.
• a number of these cell lines e.g., NIH-3T3, HeLa, NG115, CHO, HEK, 293 and COS7 can be tested to con- firm that they lack an endogenous Ca 2+ receptor.
• Those lines lacking a response to external Ca 2+ can be used to establish stably transfected cell lines expressing the cloned Ca 2+ receptor.
• Sequence analysis of Ca 2+ receptor cDNA clones iden- tified by expression cloning will delineate the open read ⁇ ing frame encoding the receptor protein.
• the coding region of the cDNA will be subcloned into multiple cloning site of the eukaryotic expression vector pMSG.
• This vec ⁇ tor allows high level transcirption driven by the mouse mammary tumor virus (MMTV) promoter, and is active in a wide variety of mammalian cells.
• the vector also contains a gpt gene for resistance to mycophenolic acid which is under the control of the SV40 early promoter, and sequences necessary for selection and growth in bacteria.
• the Ca + receptor expression construct will be introduced into cultured cells by the appropriate tech ⁇ nique, either Ca 2+ phosphate precipitation, DEAE-dextran transfection, lipofection or electroporation. Following the transfection procedure, cells are grown in the pres ⁇ ence of the antibiotic G418 to select for cells expressing the neomycin resistance gene. Colonies of G418 resistant transfectants will be subcloned and established as indivi ⁇ dual cell lines.
• Ca 2+ receptor protein in G418 resistant cells will be assessed by several meth ⁇ ods. Southern blot and slot blot analysis will confirm the presence and the copy number of the receptor cDNA sequence. Northern blot analysis will be used to demon- strate that receptor mRNA is being transcribed from the plasmid construct. Functional expression of the receptor protein will be determined by measuring the mobilization of intracellular Ca 2+ in response to externally applied Ca 2+ receptor agonists. Cloning the Ca 2+ receptor enables both structural and functional studies of this novel receptor. Recombinantly produced receptor may be crystallized for structural stu ⁇ dies. Stably transfected cell lines expressing the recep ⁇ tor can be used for high-throughput screening of natural product or other compound libraries.
• Molecules of the requisite potency and specificity can be labeled (radio- actively or fluorescently) .
• the ability of test mole ⁇ cules/extracts to displace such a labeled molecule will form the basis of a high-throughput assay for screening.
• these receptors may be cloned in a manner analogous to that described above for the para ⁇ thyroid cell calcium receptor.
• mRNA from human osteoclastoma tissue encodes the osteoclast calcium receptor ( Figure 34) .
• a clone for the human osteoclast receptor one need only isolate mRNA from osteoclastoma tissue, prepared a cDNA library and assay/ fractionate subpools as described above.
• the preferred receptors for drug screening are of human ori- gin.
• a clone encoding a calcium receptor from one species may be used to obtain the corresponding human cDNA clone by cross-hybridization as is well known by those skilled in the art.
• the clone of the parathyroid cell or other cell Ca + receptor allows isolation of genes encoding similar Ca 2+ -sensing proteins in other cells, and expression of those proteins. This is achieved by a vari ⁇ ety of approaches.
• Southern blot analysis of human geno- mic DNA utilizing the Ca 2+ receptor cDNA as a hybridiza ⁇ tion probe, will give an indication of the number of related sequences encoded within the genome; hybridization at varying stringencies will give an indication of the degree of divergence among the related sequences. This will provide information about the potential number of genes encoding related receptor proteins.
• Northern blot analysis with Ca + receptor cDNA as probe will determine if the same or related transcripts are present in various tissues. If related transcripts homologous to the para ⁇ thyroid cell Ca 2+ receptor are detected, it is a relatively simple matter to obtain clones of these mRNAs, either by screening the appropriate cDNA libraries or by polymerase chain reaction (PCR) techniques.
• PCR polymerase chain reaction
• Novel receptor clones so obtained can be assessed functionally by expression, either in oocytes or in transfected cell lines.
• Trans ⁇ fected cell lines expressing a cell-specific Ca 2+ receptor can then provide a means of high-throughput screening for molecules that act specifically on the Ca 2+ -sensing mecha- nism of, for example, osteoclasts or juxtaglomerular cells.
• the calcium receptor can be cloned by expression in eukaryotic cells.
• a cDNA library can be prepared from parathyroid mRNA and cloned into the eukaryotic expression vector, pCDNAl. Subpools from this library can be transfected into eukary ⁇ otic cells such as C0S7 or HEK293 cells resulting in rela ⁇ tively high level transient expression of encoded cDNA sequences. Cells transfected with a function calcium receptor clone will express the calcium receptor which can then be activated by calcium, neomycin or other calcimi ⁇ metic compounds.
• library subpools containing the calcium receptor are identified by their ability, upon transfection into eukaryotic cells, to induce a calcium or calcimimetic-specific increase in fluorescense.
• This fluorescense can be detected using either a fluori eter or a fluorescence activated cell sorter (FACS) .
• the Ca 2+ receptor can be cloned by use of a monoclonal antibody generated against the receptor.
• Monoclonal antibodies provide powerful tools for the immunoaffinity purification of specific proteins. Once purified, limited amino acid sequence data can be obtained from the protein of interest, and used to design oligonucleotide sequence probes to screen for clones of the complete cDNA sequence.
• hybridomas whole bovine para ⁇ thyroid gland cells are used as the immunogen. Purified, dispersed cells are obtained, and live cell preparations are injected intraperitoneally into the appropriate mouse strain, according to established procedures. Standard protocols are followed for immunization schedules and for the production of hybridomas. A two-step screening proce ⁇ dure is used to identify hybridomas secreting monoclonal antibodies that recognize the Ca 2+ receptor. The initial screen will identify those monoclonals that recognize parathyroid cell surface antigens. Immunohistochemical techniques are then used to screen hybridoma supernatants for the presence of mouse antibodies that bind to the surface of parathyroid cells. This screen can be per ⁇ formed on fixed sections of parathyroid gland tissue, or on dispersed cells in primary culture. The techniques for this assay are well established in the literature.
• This screen will identify hybridomas producing mono- clonal antibodies to a variety of cell surface determi ⁇ nants, and monoclonals specific for the Ca 2+ receptor would be expected to comprise only a small subset of these.
• hybridoma supernatants that test positive in the ini- tial screen are assayed for their ability to block the response of cultured parathyroid cells to Ca 2+ receptor agonists.
• Some antibodies that bind to the extracellular domain of the receptor are expected to inhibit or activate ligand binding or to otherwise interfere with or affect receptor activation.
• Monoclonal antibodies positive in both screens are characterized through Western blotting, immunoprecipita ⁇ tion and immunohistochemistry. This permits the determi ⁇ nation of the size of the antigen that is recognized and its tissue distribution.
• the appropriate monoclonal anti ⁇ body is then used for purification of the Ca 2+ receptor protein by immunoaffinity chromatography, following stan ⁇ dard techniques. Sufficient quantities of protein are obtained to allow limited amino acid sequence determina ⁇ tion.
• Degenerate oligonucleotide probes are then designed on the basis of the peptide sequence information. These probes are then used to screen parathyroid gland cDNA libraries for full length clones of the Ca 2+ receptor.
• Clones obtained are characterized by DNA sequencing and by functional expression in the oocyte system and in cultured mammalian cell lines.
• the antibodies can be used to screen expression libraries, e.g.. cDNA libraries in ⁇ gtll or its equivalent, to determine those clones expressing antigeni- cally reactive protein. Such clones can then be sequenced to determine whether they encode a protein that might be a Ca 2+ receptor.
• phage display libraries can be used to clone and analyze calcium receptors in place of monoclonal anti ⁇ bodies.
• antibody variable regions or random peptides are shotgun cloned into phage expression vectors such that the antibody regions or peptides are displayed on the surface of the phage particle.
• Phage which display antibody regions or peptides capable of high specific binding to calcium receptors will bind to cells which display these receptors (e.g. parathyroid cells, C-cells, osteoclasts, etc.). Millions of such phage can be panned against these cell types selecting only those phage which can bind to these cells (which includes those phage binding to calcium receptors) .
• the screens described above for monoclonal anti- bodies can be used to isolate phage which display calcium receptor-binding antibody or peptide regions, and these phage can be used to isolate the calcium receptor for pur ⁇ poses of structural identification and cloning.
• Kits to prepare such phage display libraries are commercially available (e.g. Stratacyte, or Cambridge Antibody Tech ⁇ nology Limited) .
• Recombinant phage endowed with such calcium receptor-binding properties can also be used in lieu of monoclonal antibodies in the various analyses of calcium receptors.
• Such phage can also be used in high throughput binding competition screens to identify organic compounds capable of functional binding to calcium recep ⁇ tors which can serve as structural leads for the develop ⁇ ment of human therapeutics acting at the calcium receptor.
• affinity cross-linking of radioligands to their receptors can be used to isolate the receptor protein as described by Pilch & Czech, 1 Receptor Biochem. Methodol. 161, 1984. Covalent attachment of a radioligand allows extensive washing to remove non ⁇ specific binding.
• a high affinity molecule e.g...
• Protamines because of their much smaller size, may be preferable in cross-linking studies and can be reductively alkylated as described by Dottavio- Martin & Ravel, 87 Analvt. Biochem. 562, 1978.
• Nonspecific labelling is kept to a minimum by cross- linking in the presence of unlabeled polycations and di- and trivalent cations. At high concentrations of these molecules nonspecific interactions of the label with the cell surface might be reduced.
• HPT Primary hyperparathyroidism
• One of the major defects in HPT appears to be a diminished sensitivity of parathyroid cells to negative feedback regulation by extracellular Ca 2+ .
• the "set-point" for extracellu ⁇ lar Ca 2+ is shifted to the right so that higher than normal concentrations of extracellular Ca 2+ are required to depress PTH secretion.
• primary HPT even high concentrations of extracellular Ca 2+ often depress PTH secretion only partially.
• secondary (uremic) HPT a similar increase in the set-point for extracellular Ca 2+ is observed even though the degree to which Ca 2+ suppresses PTH secretion is normal.
• the Ca 2+ receptor constitutes a discrete molecular entity for pharmacological intervention.
• Molecules that mimic or antagonize the action of extracellular Ca 2+ are beneficial in the long-term management of both primary and secondary HPT.
• Such molecules provide the added impetus required to suppress PTH secretion which the hypercalcemic condition alone cannot achieve.
• Such molecules with greater efficacy than extracellular Ca 2+ may overcome the apparent nonsuppressible component of PTH secretion which is particularly troublesome in adenomatous tissue.
• Calcitonin secretion from parafollicular cells in the thyroid is regulated by changes in the concentration of extracellular Ca 2+ .
• Renin secretion from juxtaglomerular cells in the kidney is depressed by increased concentrations of extracellular Ca 2+ .
• Extracellular Ca 2+ causes the mobilization of intra ⁇ cellular Ca 2+ in these cells.
• Isolated osteoclasts respond to increases in the concentration of extracellular Ca 2+ with corresponding increases in [Ca 2+ ]j that arise partly from the mobilization of intracellular Ca 2+ .
• Ca 2+ receptor on parathyroid cells and other cells will allow the presence of homologous proteins in other cells to be directly assesed.
• a family of struc ⁇ turally homologous Ca 2+ receptor proteins can thus be obtained.
• Such receptors will allow understanding of how these cells detect extracellular Ca 2+ and enable evaluation of the mechanism(s) as a site of action for the therapeu ⁇ tics described herein effective in the treatment of HPT, osteoporosis, and hypertension, and novel therapies for other bone and mineral-related diseases.
• recom- binant Ca 2+ receptor proteins may be used in therapy, and introduced by standard methods, e.g.. by transfection of nucleic acid encoding that protein.
• such protein is useful in assays for calcimimetic molecules of this invention.
• Example 1 Screening Calcimimetic Molecules on Bovine Parathyroid cells
• Dissociated bovine parathyroid cells were purified on gradients of Percoll and cultured overnight in serum-free medium. The cells were subsequently loaded with fura-2 and the concentration of free intracellular Ca 2+ measured fluorimetricly. Changes in [Ca 2+ ] j were used to screen for molecules active at the Ca 2+ receptor.
• a molecule was required to show the normal effects caused by increasing extracellular Ca 2+ and trig ⁇ gered by the activation of the Ca 2+ receptor. That is,
• the molecule must cause a decrease in iso- proterenol-stimulated cyclic AMP formation which is blocked by pertussis toxin; 3) The molecule must inhibit PTH secretion over the same range of concentrations that cause the increase in [Ca 2+ ] £ ; and
• spermine, neomycin B, and protamine evoked rapid and transient increases in [Ca 2+ ]j in fura-2-loaded bovine parathyroid cells (Figs. 6, 7, 11) . They did not, however, cause sustained, steady- state increases in [Ca 2+ ]; in bovine parathyroid cells (Fig. 6, 11), although they did in human parathyroid cells (Fig. 19) . In this respect, they resembled the cytosolic Ca 2+ response elicited by extracellular Mg 2"1" , which causes the mobilization of intracellular Ca 2+ unaccompanied by an influx of extracellular Ca 2+ in bovine cells (Fig. lib) .
• SUBSTITUTESHEET transient increases in [Ca 2+ ] j evoked by spermine (Fig. 6) .
• a maximally effective concentration of sper ⁇ mine or neomycin B (Fig. 12) blocked transient, but not steady-state increases in [Ca 2+ ]i evoked by extracellular Ca 2+ .
• Mobilization of intracellular Ca 2+ is the essential mechanism associ ⁇ ated with the inhibition of PTH secretion. This is impor- tant because it defines the sufficient mechanism to be affected if a molecule is to affect PTH secretion; mole ⁇ cules stimulating selectively the influx of extracellular Ca 2+ will be relatively ineffective in suppressing PTH secretion. In contrast, molecules causing solely the mobilization of intracellular Ca 2+ should be just as efficacious as extracellular Ca 2+ in suppressing PTH secretion.
• Protamine Pertussis toxin blocks the inhibitory effects of extracellular Ca 2+ and molecular polycations on cyclic AMP formation.
• Bovine parathyroid cells were cultured for 16 h with or without 100 ng/ml pertussis toxin. The cells were subsequently washed and incubated for 15 min with 10 ⁇ M isoproterenol with or without the indicated concentra ⁇ tions of extracellular Ca 2+ or molecular polycations.
• Total cyclic AMP (cells + supernatant) was determined by RIA and the results are expressed as a percentage of the levels obtained in 0.5 mM Ca 2+ (112 ⁇ 17 pmole/10 6 cells). Each value is the mean ⁇ SEM of three experiments.
• Neomycin B caused not only a transient but additionally a steady-state increase in [Ca 2+ ] £ in human parathyroid cells (Fig. 19a) .
• the pattern of change in [Ca 2+ ] £ evoked by extracellular Ca 2+ , Mg 2+ or neomycin B is very similar.
• Neomycin B Cytosolic Ca + transients elicited by neomycin B persisted in the presence of La 3+ (l ⁇ M) and absence of extracellular Ca 2+ .
• Neomycin B therefore causes the mobilization of intracellular Ca 2+ in human parathyroid cells.
• Neomycin B inhibited PTH secretion from human parathyroid cells at concentrations that caused the mobil ⁇ ization of intracellular Ca + (Fig. 13) .
• bovine cells can be used for initial studies to screen test molecules for activity, it is important to perform follow-up studies using human para ⁇ thyroid cells.
• a neoplastic cell line derived from a rat medul ⁇ lary thyroid carcinoma (rMTC 6-23 cells) was used. Both spermine (10 mM) and neomycin B (5 mM) were without effect on basal [Ca 2+ ] ; in these cells. Nor did either molecule affect the response to the subsequent addition of extra ⁇ cellular Ca 2+ . Representative results documenting the lack of effect of neomycin B are shown in Fig 21.
• neomycin B (5 mM) was without effect on basal [Ca 2+ ]; and did not affect increases in [Ca 2+ ] ; elicited by extracellular Ca + .
• the overall picture that develops from these studies is that spermine and neo- ycin B are without effect on basal or stimulated levels of cytosolic Ca 2+ in osteoclasts.
• the failure of the molecular polycations to affect the Ca 2+ -sensing mechanisms of C-cells or osteoclasts demonstrates the ability to discover or design novel lead molecules that act specifically on the parathyroid cell Ca 2+ receptor or otherwise modulate one or more functions of the parathyroid cell's normal response to [Ca 2+ ] .
• Putrescine (+2) and cadaverine (+2) were inactive at a concentration of 2mM.
• Another straight-chain polyamine, DADD behaved some ⁇ what differently from the other polyamines and is described in Example 7.
• Kanamycin (+4.5) and lincomycin (+1) were without effect at a concentration of 500 ⁇ M. Within the aminoglycoside series, there is a correlation between net positive charge and potency. However, neomycin is considerably more potent than various polyamines (NPS 381, NPS 382, NPS 383, PEHA) that have an equal or greater positive charge.
• Protamine and polymers of lysine or arginine varying in peptide length were screened for their ability to mobilize intracellular Ca + as in Example l. The resulting
• Molecules selected from the class of arylalkylamine toxins derived from the venoms of wasps and spiders were screened as in Example 1.
• Philanthotoxin-433 (+3) was without effect at a con ⁇ centration of 500 ⁇ M. It is similar in structure to the argiotoxins described below.
• Argiotoxin-636 (400 ⁇ M) did not elicit increases in [Ca 2+ ]; but it did potentiate cytosolic Ca 2+ responses to the subsequent addition of extracellular Ca 2+ . This is a feature common to all molecules that activate the Ca 2+ receptor and is also seen with a variety of extracellular divalent cations. This is considered in more detail in Example 7.
• argiotoxin-636 argiotoxin-659 elic ⁇ ited increases in [Ca 2+ ]; with an EC J0 of 300 ⁇ M.
• Argiotoxin- 659 differs from argiotoxin-636 in having a hydroxylated indole moiety rather than a dihydroxyphenyl group. This • is the only difference in the structure of these two molecules. Thus, the difference in potency lies in the nature of the aromatic group, not in the polyamine chain which carries the positive charge.
• Ca 2+ channel blockers i.e.. those molecules which block influx of extracellular Ca 2+ through voltage- sensitive Ca 2+ channels, were screened as in Example 1.
• the phenylalkylamines examined were verapamil, D-600 (a methoxy-derivative of verapamil), TMB-8, and an analog of TMB-8, NPS 384.
• the first three molecules were tested at a concentration of 100 ⁇ M.
• the phenylalkylamines behaved differently from other molecules examined. They evoked no change in [Ca 2+ ]i when added to cells bathed in buffer containing a low concentration of extracellular Ca 2+ (0.5 mM) .
• verapamil, D-600, and TMB-8 potenti ⁇ ated the mobilization of intracellular Ca 2+ elicited by extracellular divalent cations and they additionally blocked the influx of extracellular Ca 2+ .
• these molecules were capable of evoking a small but robust increase in [Ca 2+ ] j that arose from the mobilization of intracellular Ca 2+ .
• the phenylalkylamines act differently than organic polycations like neomycin.
• the data suggest that vera- pamil, D-600 and TMB-8 are partial agonists at the Ca 2+ receptor, in contrast to the other molecules examined which are full agonists.
• Molecule NPS 384 at a concentration of 300 ⁇ M, did not evoke an increase in [Ca 2+ ] ; but it blocked influx of extracellular Ca + . Testing at higher concentrations may reveal an ability of this molecule to cause the mobiliza ⁇ tion of intracellular Ca 2+ .
• [Ca 2+ ]j (arising from intracellular Ca 2+ mobilization) that is important.
• [Ca 2+ ]i in parathyroid cells shows that transient increases in [Ca 2+ ] ; almost invariably result from the mobilization of intracellular Ca 2+ and therefore reflects activation of the Ca 2+ receptor.
• the benzothiazipine examined, diltiazem was similar in all respects to verapamil and D-600 and was also effec ⁇ tive at 100 ⁇ M.
• TMB-8 potentiated transient increases in [Ca 2+ ] ; at 100 ⁇ M whereas NPS 384 fails to do so even at 300 ⁇ M, yet these molecules carry the same net positive charge. It follows that some other structural feature, unrelated to net charge, imparts greater potency to TMB-8.
• Neomycin on the other hand, evoked a large increase in [Ca 2+ ]j in human parathyroid cells when tested at a con ⁇ centration of 20 ⁇ M.
• the magnitude of the response elici- ted by neomycin was equal to that evoked by a maximally effective concentration of extracellular Ca 2+ .
• Oocytes injected with mRNA from human parathyroid cells express the Ca 2+ receptor and mobilize intracellular Ca 2+ in response to a variety of extracellular inorganic
• oocytes were injected with bovine para ⁇ thyroid mRNA or water and, after 72 hours exposed to serum or 10 mM neomycin. Prior to being stimulated, oocytes were loaded with 45 Ca 2+ . Stimulation with serum for 20 min resulted in intracellular 45 Ca 2+ release representing a 45% increase compared to mock challenge with buffer. Chal ⁇ lenge with 10 mM neomycin for 20 min. resulted in a 76% increase in 45 Ca 2+ release.
• the assay is sensitive enough for use in cloning the Ca 2+ receptor, and has the advantage of a higher throughout than the electrophysiological mea ⁇ surement of Ca + activated Cl" current.
• human osteoclastoma tissue was obtained from bone biopsy tissue. Oocytes injected with mRNA isolated from this tissue were challenged with 30 mM Ca 2+ . Controls did not respond while 8 of 12 oocytes injected with osteoclastoma mRNA responded appropriately (Fig. 34) .
• These experiments provide the first evidence that the Ca 2+ response of osteoclasts to extracellular Ca 2+ is in fact genetically encoded. The results also indicate that the osteoclast Ca 2+ receptor may be cloned by expres ⁇ sion in Xenopus oocytes.
• Example 10 Molecule Screening on Rat Osteoclasts
• the different sensitivities of parathyroid cells and osteoclasts to extracellular Ca 2+ suggest that their Ca 2+ receptors are different. While parathyroid cells respond to extracellular Ca 2+ concentrations between 0.5 and 3 mM, osteoclasts respond only when the level of extracellular Ca 2+ increases beyond 5 mM. This rather high concentration of Ca 2+ is nonetheless physiological for osteoclasts; as they resorb bone, the local concentration of extracellular Ca 2+ may reach levels as high as 30 mM.
• Osteoclasts were obtained from the long bones of neonatal rats. [Ca 2+ ]j was measured in single cells using the fluorimetric indicator indo-1.spermine, spermidine, neomycin, and verapamil were tested, and none of these caused any large increase in [Ca 2+ ] j in osteoclasts (although small responses were detected) . At a concentration of 1 mM, spermidine caused a small increase in [Ca 2+ ]j (about 10% of that evoked by a maximal concentration of extracellular Ca 2+ ) .
• Neomycin (10 mM) norspermine (10 or 20 mM) caused increases in [Ca 2+ ]j in rat osteoclasts.
• Neomycin (10 mM) did not block the increase in [Ca 2+ ]; elicited by the subsequent addition of 25 mM extracellular Ca 2+ .
• Pretreatment with spermine (20 mM) did depress the response to extracellular Ca 2+ .
• Verapamil (100 ⁇ M) caused no detectable increase in [Ca 2+ ] ; but it did block the response to extracellular Ca 2+ .
• Comparisons between osteoclasts and parathyroid cells show that molecules active on the latter are relatively ineffective in osteoclasts. This demonstrates that drugs that target a specific Ca 2+ receptor without affecting those receptor types present on other Ca 2+ -sensing cells are readily developed. Similarly, drugs active at two or more such Ca 2+ receptors may also be developed.
• the parathyroid cell Ca 2+ receptor senses levels of extracellular Ca 2+ around 1.5 mM whereas the Ca 2+ receptor on the osteoclast responds to levels around 10 mM (Fig. 22) .
• Neomycin or spermine which activate the parathyroid cell Ca 2+ recep ⁇ tor, fail to affect the Ca 2+ receptors on C-cells or osteo ⁇ clasts (Figs. 21 and 23) .
• the lead molecules presently under development will affect selec ⁇ tively the activity of a specific type of Ca 2+ -sensing cell in vivo.
• Bovine parathyroid cells and C-cells were loaded with fura-2, and rat osteoclasts with indo-1 and the potency of the indicated molecules to mobilize intracellular Ca 2+ determined by constructing cumulative concentration-response curves .
• Molecules listed as "inactive” did not alter [Ca 2+ ]j when tested at a concentration of 1 mM.
• NPS 456 is a potent activator of the parathyroid cell Ca 2+ receptor. This molecule is notable because it possess only one positive charge yet is much more potent than many polybasic molecules. Brief (2 min) pretreatment with PMA shifts the concentration- response curve for NPS 456 to the right. This indicates that NPS 456 acts through the same mechanism used by extracellular Ca 2+ . NPS 456 evokes the mobilization of intracellular Ca 2+ in Xenopus oocytes expressing the
• NPS 456 contains a chiral carbon, and therefore exists in two isomeric forms. Both isomers have been synthesized and examined for activity.
• the R-isomer, NPS 447 is 12-times more potent than the S-isomer, NPS 448 (Fig. 28) . This is the first demonstration that a Ca 2+ receptor can recognize an organic molecule in a stereospecific manner.
• NPS 447 is a structurally simple molecule with selective and potent effects on the parathyroid cell Ca 2+ receptor
• structure-activity studies around this lead molecule are simple. The aim of these studies is to generate an array of related molecules with various char ⁇ acteristics from which the final development candidate can be selected.
• This effort has already revealed some of the structural domains of NPS 447 that contribute to activity and potency.
• the novel compound NPS 459 is an analog of NPS 447 that is smaller (MW ⁇ 240) yet nearly as potent as the parent molecule, whereas several other analogs are relatively inactive.
• the most interesting molecules from this analog project can be put into in vivo testing for effects on PTH secretion and serum Ca 2+ levels (see Examples 15, 16, 17, 18 and 23).
• the novel compound NPS 467 is an even smaller mole- cule than NPS 447 yet the former is about 3-fold more potent than the latter in causing the mobilization of intracellular Ca 2+ in parathyroid cells.
• NPS 467 is a racemic mixture. It is anticipated that Resolu ⁇ tion of NPS 467 into its enantiomers provides an isomer of even greater potency than the racemic mixture (see Example 16) .
• NPS 551 is another novel compound as potent as NPS 467 in causing the mobilization of intracellular Ca 2+ in parathyroid cells.
• NPS 551 is a racemic mixture and it is anticipated that the resolution of NPS 551 into its enan- tiomers will result in an isomer that is more potent than the racemic mixture. Further structure-activity studies on molecules related to NPS 447, NPS 467, NPS 551 and NPS 568 are expected to yield pure isomers with greater potency than these molecules in their racemate forms.
• NPS 456 is the most potent molecule activate on Xenopus oocytes expressing the parathyroid cell Ca 2+ receptor.
• the results obtained in this expres ⁇ sion system with neomycin and NPS 456 demonstrate that these molecules act directly on the Ca 2+ receptor.
• the strategy used for elucidating the mechanism of action of extracellular Ca 2+ on the osteoclast was similar to that proven effective in parathyroid cells.
• the first experiments examined the effects of La 3+ on [Ca + ] ; in single rat osteoclasts loaded with the fluorimetric indicator indo-1. As described above, trivalent cations like La 3+ are impermeant and block Ca 2+ influx. Low micromolar concentrations of La 3+ partially depressed extracellular Ca 2+ -induced increases in [Ca 2+ ]j (Fig. 29) .
• the demonstration of a La 3+ -resistant increase in [Ca 2+ ]i provides evidence for the mobilization of intracellular Ca 2+ .
• the results of these experiments parallel those obtained in parathyroid cells and suggest that similar mechanisms are used by extracellular Ca 2+ to regulate [Ca 2+ ]j in both cell types.
• results obtained with a variety of di- and tri ⁇ valent cations are all consistent with the presence of a Ca 2+ receptor on the surface of the osteoclast that is coupled to the mobilization of intracellular Ca 2+ and influx of extracellular Ca 2+ through voltage-insensitive channels.
• Results show evidence for genetic material in human osteoclasts that encodes a Ca + receptor protein (see below) .
• Transient increases in [Ca 2+ ]i resulting from the mobilization of intracellular Ca 2+ are sufficient to inhibit osteoclastic bone resorption in vitro.
• activation of the Ca 2+ receptor appears to be a viable means of inhibiting the activity of osteoclasts. ?
• NPS 449 is presently the lead molecule for calcimi ⁇ metic drugs on this receptor. It is a small molecule (MW ⁇ 425) and it mobilizes intracellular Ca 2+ in rat osteo- clasts with a EC, of 200 ⁇ M (Figs. 31A and 3IB) . Although the potency of NPS 449 is relatively low, it has a simple structure with only one positive charge and is expected to have desirable pharmacodynamic and pharmacokinetic properties. NPS 449 was examined for its ability to inhibit bone resorption in vitro. This was done by morphometric analy ⁇ sis of pit formation on thin slices of bovine cortical bone using scanning electron microscopy.
• Rat osteoclasts were incubated for 24 hours in slices of bone in the pres- ence or absence of various concentrations of NPS 449.
• NPS 449 caused a concentration-dependent inhibition of bone resorption with an ICso of 10 ⁇ M.
• the anticipated results provide the first demonstration that molecules acting at this novel site can inhibit osteoclastic bone resorption.
• More potent analogs of NPS 449 will be generated using synthetic chemistry and will be tested and assayed using the methods described herein.
• Calcimimetic drugs selectively affecting C-cells are useful in the treatment of osteoporosis.
• the mobilization of intracellular Ca 2+ is used as a functional index of Ca 2+ receptor activity.
• the screening effort in C-cells is facilitated by the availability of cultured cell lines expressing the C-cell phenotype (e.g.. rat medullary thyroid carcinoma cells; rMTC 6-23 cells) .
• C-cell phenotype e.g.. rat medullary thyroid carcinoma cells; rMTC 6-23 cells
• Selected for initial study were three arylalkylamine molecules. Two are naturally occurring (agatoxin 489 and agatoxin 505) and the other (NPS 019) is a synthetic agatoxin analog.
• Agatoxin 505 was found to block extra ⁇ cellular Ca 2+ -induced increases in [Ca 2+ ] £ , with an IC 50 of 3 ⁇ M.
• agatoxin 489 was found to mobilize intracellular Ca 2+ in rMTC cells with an EC*, of 150 ⁇ M. This was the first organic molecule discovered that was found to activate the C-cell Ca 2+ receptor.
• the synthetic analog, NPS 019 was even more potent and mobilized intra- cellular Ca 2+ with an EC JO of 5 ⁇ M (Fig. 32) . It is sig ⁇ nificant that the only structural difference between NPS 019 and agatoxin 489 is the presence or absence of an hydroxyl group. The fact that such subtle differences in structure affect profoundly the potency of molecules indi- cates a structurally specific binding site on the Ca 2+ receptor. This, in turn, encourages the view that very potent and selective activators of Ca 2+ receptors can be developed.
• NPS 019 which is a small molecule (MW ⁇ 500), is a lead molecule for development of calcimimetics of the C-cell Ca 2+ receptor and can be tested for its ability to stimulate calcitonin secretion in vitro. Subsequent in vivo testing will then determine the ability of this molecule to stimulate calcitonin secretion and inhibit bone resorption. These in vivo studies will be performed in rats. The results obtained in these studies, which are anticipated to be positive, will provide the first evi-
• SUBS TITUTESHEET dence showing that a small organic molecule acting on a novel receptor can stimulate calcitonin secretion and depress bone resorption.
• NPS 021 For a compound to be considered a calcilytic, it must block the effects of extracellular Ca 2+ or a calcimimetic compound on an extracellular Ca 2+ -sensing cell.
• An example of a calcilytic compound is NPS 021, the structure of which is provided in Fig. 1.
• NPS 021 blocks increases in [Ca 2+ ]; elicited by extracellular Ca 2+ .
• the IC ⁇ , of NPS 021 for blocking this response is about 200 ⁇ M and, at concentra ⁇ tions around 500 ⁇ M, the increase in [Ca + ]j evoked by extracellular Ca 2+ is abolished.
• NPS 021 does not by itself cause any change in [Ca 2+ ]j when tested at low [Ca 2+ ] (0.5 mM; Fig. 37).
• NPS 467 at doses effective in lowering blood Ca 2+ , caused a dose-dependent reduction in circulating levels of PTH (Fig. 39) .
• the hypocalcemic effect of NPS 467 was maximal within three hours and returned toward control levels after 24 hours (Fig. 40) .
• NPS 467 (the EE isomer; see Example 16) was also effective in lowering serum ionized Ca 2+ in rats maintained on a normal, calcium-replete diet.
• NPS 467 (EE isomer, 10 mg/kg i.p.) caused a rapid fall in serum levels of ionized Ca 2+ which were maximal by 1 hr (22%) decrease from the control level) and remained depressed at or near this level for up to 6 hours.
• NPS 467 is a racemic mixture. Resolution of NPS 467 into its two enantiomers was achieved by separation on a chiral column. The EE-isomer (for "early eluting", see Example 21) was about 100-fold more potent than the LE- isomer (for "late-eluting") in activating the bovine parathyroid cell Ca 2+ receptor m vitro as assessed by the ability of the enantiomers to evoke increases in the [Ca 2+ ] ; in parathyroid cells (Fig. 41) .
• NPS 467 The isomers of NPS 467 were examined for effects on serum Ca 2+ as in Example 15. Consistent with the in vitro results, the EE-isomer or NPS 467 proved to be more potent than the LE-isomer in lowering serum Ca 2+ in vivo (Fig. 42; each compound was tested at a concentration of 5 mg/kg body weight) .
• Example 17 NPS 467 Lowers Serum Ionized CaJcium in an in vivo Model of Secondary Hyperparathyroidism
• NPS 467 EE isomer; 10 mg/kg i.p.
• the administration of NPS 467 caused a rapid (within 2 hours) fall in serum ionized Ca 2+ levels to 83% of controls in an animal model of secondary hyperparathyroidism. This suggests that com ⁇ pounds of this sort will effectively depress PTH secretion in patients with secondary hyperparathyroidism and hyper- plastic parathyroid glands.
• Example 18 NPS 467 Fails to Lower Serum Ionized -_ «]-_.UT ⁇ Levels in Parathyroidectomized Animals
• NPS 467 acts to cause a hypocalcemic response
• the parathyroid glands in rats were surgically removed. Animals receiving a total parathyroidectomy become hypocalcemic and are largely dependent upon dietary calcium to maintain serum Ca 2+ homeostasis. Parathyroidectomized animals had serum ionized Ca 2+ levels of 0.92 mM which fell gradually to 0.76 mM after 6 hours of fasting.
• the administration of a single dose of NPS 467 EE (10 mg/kg i.p.) did not cause any change in serum ionized Ca 2+ levels over a period of 6 hours.
• NPS 467 EE can target the parathyroid glands in vivo. The results are consistent with the view that NPS 467 EE acts on the parathyroid cell Ca 2+ receptor in vivo to depress secretion of PTH and thereby cause serum levels of ionized Ca 2+ to fall.
• Dissociated parathyroid cells were prepared from a parathyroid adenoma obtained by surgery from a patient with primary hyperparathyroidism. The cells were loaded with fura-2 and [Ca 2+ ] j measured as described above. Both NPS 467 EE and NPS 568 EE caused concentration-dependent increase in [Ca 2+ ] £ . The ECso's for NPS 467 EE and NPS 568 EE were 20 and 3 ⁇ M, respectively. Both these compounds are thus able to increase [Ca 2+ ] j in pathological human tissue and would thus be expected to decrease serum levels of PTH and Ca 2+ in patients with primary hyperpara ⁇ thyroidism.
• NPS 467 EE Dissociated bovine parathyroid cells were used to further explore the mechanism of action of NPS 467 at the receptor level.
• NPS 467 EE caused a rapid and transient increase in [Ca 2+ ]i which persisted in the presence of 1 ⁇ M La 3+ and was partially depressed by pretreatment with PMA (100 nM for 2 min.).
• NPS 467 (EE isomer, 30 ⁇ M) caused a rapid increase in Cl" conductance in Xenopus oocytes injected with parathyroid cell mRNA. All these results are consistent with an action of NPS 467 on the Ca 2+ receptor.
• Neomycin was used because it mimics, in nearly all respects, the effects of extracellular Ca 2+ on parathyroid cells and on Xenopus oocytes expressing the parathyroid cell Ca 2+ receptor.
• the addition of 10 ⁇ M neomycin did not by itself cause an increase in [Ca 2+ ] £ under these conditions.
• NPS 467 EE (30 ⁇ M) now elicited a transient increase in [Ca 2+ ]i which, because there was no extracellular Ca 2+ present, must have come from the mobilization of intracel ⁇ lular Ca 2+ .
• NPS 467 EE 30 ⁇ M
• NPS 467 This concentration of NPS 467 is maximally effective in increasing [Ca 2+ ]j when extracellular Ca 2+ (0.5 mM) is present. However, the subsequent addition of 10 ⁇ M neo ⁇ mycin now evoked a transient increase in [Ca + ]i. Presum- ably, neomycin binds to the same site as extracellular Ca 2+ and can functionally substitute for it. Using a submaxi- mal concentration, which by itself causes no response, achieves partial occupancy of the Ca 2+ -binding site and allows activation of the Ca 2+ receptor by NPS 467. Additional studies to further define the mechanism of action of NPS 467 were performed.
• NPS 467 EE did not cause any increase in [Ca 2+ ] £ . It did not, however, affect the increase in [Ca 2+ ] j evoked by the sub ⁇ sequent addition of 100 ⁇ M neomycin.
• Ligand binding to the former site can result in full activation of the Ca 2+ receptor whereas ligand binding to the latter site can only occur and/or be functionally relevant when the extra ⁇ cellular Ca 2+ -binding site is occupied to some as yet undefined degree. It is possible that ligand binding to the extracellular Ca 2+ -binding site exposes a previously occluded binding site for NPS 467. It appears that the NPS 467-binding site is an allosteric site that augments receptor activation in response to ligand binding at the extracellular Ca 2+ -binding site. The data demonstrate that the parathyroid cell Ca 2+ receptor possesses at least two distinct sites for organic ligands.
• NPS 467 defines a previously unrecognized binding site on the Ca 2+ receptor. Binding to this site can only occur and/or results in full activation of the Ca 2+ receptor when the extracellular Ca 2+ -binding site is partially occupied. Ligands acting at either site are effective in suppressing serum Ca + levels n vivo.
• reaction mixture was chromatographed through silica (48 x 4.6 cm) using a gradient of chloroform- methanol-isopropylamine (99:1:0.1) to (90:10:0.1) which yielded 13.66 g of purified NPS 467.
• This material was dissolved in Hexane-isopropanol (99:1) containing 0.1% diethyla ine to yield a solution with a concentration of 50 mg/ml.
• Chiral resolution was accomplished by chroma- tography of 4 ml of this solution (200 mg, maximum to achieve separation) through ChiralCel OD (25 x 2 cm) using 0.7% isopropylamine, 0.07% diethylamine in hexane at 100 ml/min, monitoring optical density at 260 nm. Under these conditions (with injections of 100 mg material) the early eluting isomer (NPS 467EE) began to emerge from the column at " 26 min, the late eluting isomer (NPS 467LE) began to emerge at "34 minutes. Baseline resolution was accom ⁇ plished with under these conditions.
• Each optical isomer (free base) was converted to the corresponding hydrochlor- ide salt by dissolving 3 g of the free base in 100 ml ethanol and treating with it with 100 ml water containing 10 molar equivalents HCI. Lyophilization of this solution yielded a white solid.
• NPS 467 (EE isomer) was suspended in corn oil and administered as a single oral dose through a gavage needle. Three hours later a sample of blood was taken from the tail vein and assessed for ionized Ca 2+ levels. Fig. 44 shows that NPS 467 EE caused a dose-dependent reduction in serum levels of ionized Ca 2+ when administered orally.

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