NZ330376A - Androstane steroid ligands as neurochemical initiators of change in human hypothalamic and autonomic function - Google Patents

Description

New Zealand No 330376 International No PCT/ TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates 15 06 1993, Complete Specification Filed 28 09 1993 Classification (6) A61K31/56.565 Publication date 29 September 1999 Journal No 1444 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention Androstane steroids as neurochemical initiators of change in human hypothalamic function and related pharmaceutical compositions Name, address and nationality of applicant(s) as in international application form PHERIN CORPORATION, Suite 240, 535 Middlefield Road, Menlo Park, California 94025, United States of America ^ ' ■> \J %) w 0 Under the provisions of Regulation 23 (1) Specification has been ante-dated to 19 CTb ,%* a. a NEW ZEALAND PATENTS ACT, 1953 No Date Initials Divided out ofNZ 257218 dated 28 September 1993 COMPLETE SPECIFICATION ANDROSTANE STEROIDS AS NEUROCHEMICAL INITIATORS OF CHANGE IN HUMAN HYPOTHALAMIC FUNCTION AND RELATED PHARMACEUTICAL COMPOSITIONS We, PHERIN CORPORATION, of Suite 240, 535 Middlefield Road, Menlo Park, California 94025, United States of America, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement (followed by page la) n^LLECTUAL'TOPSTYOFFicEl OF NZ j 1 3 AUG 1999 ^Received la ANDROSTANE STEROIDS AS NEUROCHEMICAL INITIATORS OF CHANGE IN HUMAN HYPOTHALAMIC FUNCTION AND RELATED PHARMACEUTICAL COMPOSITIONS BACKGROUND Technical Field This invention relates generally the use of pharmaceutical compositions for effectuating change in human hypothalamic function, thereby altering certain behavior and physiology 10 mediated by the hypothalamus of individuals. More particularly, the invention relates to the use of certain Androstene steroids as neurochemical effectuators of physiology and behavior.

Description of the Related Art 15 The present invention relates to the use of certain compounds, namely Androstane steroids, particularly Androstene steroids and related compounds as will be described herein, and the use of these compounds as human semiochemicals in order to alter 2 0 hypothalamic function, thereby affecting certain consequent behavior and physiology, e.g , the reduction of anxiety. Androstane steroids are typified by testosterone and they are characterized by a four ring steroidal structure, a methylation at IN ':HLLEC 7UA L_*R?OPE:RTV^FRCEl of W Z 1 3 AUG 1999 the 13-position and at the 10-position. Androstenes are a subset of Androstanes and have at least one double bond. Ohloff, G. et al (Helv Chim Acta (1983) 66.: 192-217) , which is incorporated herein by 5 reference, have shown that several members of this group of steroids have an odor which varies with different isomeric, diastereomeric, and enantiomeric forms. Some members of this group have been reported to act as a pheromone m some mammalian species - for 10 instance, 5a-androst-16-en-3-one and 5a-androst-16-en-3a-ol in pigs (Melrose, D.R., et al . Br vet J (1971) 127 497-502). These 16-Androstenes produced by the boar induce mating behavior in estrus sows (Claus, et al . Experlmentia (1979) 35•1674-1675). 15 Some studies have noted that, in some species, various characteristics of certain 16-Androstenes (including 5a-Androst-16-en-3a-ol and 5a-Androst-16-en-3-one), such as concentration, metabolism, and localization, are sexually dimorphic 20 (BrooksbanJc et al. . J. Endocr. (1972) 52: 239-251; Claus, et al . J Endocr. (197 6) .68:4 83-484; Kwan, et al.. Med Sci Res (1987) 15.1443-1444). For instance, 5a-Androst-16-en-3a-ol and 5a-Androst-16-en-3-one, as well as Androsta-4,l6-dien-3-one, have 25 been found at different concentrations in the peripheral blood, saliva and axillary secretions of men and of women (Kwan, T.K., et a 1 . Med Sci Res. (1987) 15:1443-1444), and their function as a human pheromone, to the extent of affecting choice and 30 judgement, has been suggested (Id ; see also Gower, et al . "The Significance of Odorous Steroids in Axillary Odour", In, Perfumery, pp. 68-72, Van Toller and Dodd, Eds., Chapman and Hall, 1988); Kirk-Smith, D.A , et al . Res. Comn Psychol. Psychiat. Behav. 35 (1978) .3 :379). Androstenol (5a-androst-l6-en-3a-ol) 7 cstt -3- *\ ~ " ' 1 ' ' ^ has been claimed to exhibit a pheromone-like activity in a commercial men's cologne and women's perfume (Andron" for men and Andron" for women by Jovan). Japanese Kokai No. 2295916, refers to perfume 5 compositions containing androstenol and/or its analogues. 5a-Androstadien-3/3-ol (and perhaps the 3a-ol) has also been identified m human axillary secretion (Gower, et al , Supra at 57-60. On the other hand, there is little agreement in the 10 literature as to whether or not any putative pheromone actually plays any role in the sexual or reproductive behavior of mammals, particularly of humans See: Beauchamp, G K., et al.. " The Pheromone Concept m Mammalian Chemical 15 Communication: A Critique", In Mammalian Olfaction. Reproductive Processes and Behavior. Doty, R.L., Ed., Academic Press, 1976). See also Gower, et al.. supra at 68-73.

Described but not claimed is 20 the non-systemic, nasal administration of certain Androstane and Androstene steroids to affect a specific behavioral or physiological response in human subjects, e.g., a reduction of negative affect, mood, and character traits. In particular, nasal 25 administration provides for contacting neurochemical receptors of a heretofore poorly understood neuroendocrine structure, commonly known as the vomeronasal organ ("VNO"; also known as "Jacobson's organ"), with one or more steroid(s) or with 30 compositions containing the steroid(s). This organ is accessed through the nostrils of most higher animals - from snakes to humans, and has been associated, inter alia, with pheromone reception in certain species (see generally Muller-Schwarze & 35 Silverstem, Chemical Signals. Plenum Press, New York intellectual property office of NZ * 0 JUL 1999 Drnn. . — (1980)) The axons of the neuroepithelia of the vomeronasal organ, located supra palatmal, form the vomeronasal nerve and have direct synaptic connection to the accessory olfactory bulb and indirect input 5 from there to the cortico-medial amygdaloid basal forebrain and hypothalamic nuclei of the brain. The distal axons of termmalis nerve neurons may also serve as neurochemical receptors m the VNO.

Stensaas, L.J., et al . J. Steroid Biochem. and 10 Molec Biol. (1991) 29.:553. This nerve has direct synaptic connection with the hypothalamus.

Johnson, A et al (J Otolaryngology (1985) JL4:71-79) report evidence for the presence of the vomeronasal organ in most adult humans, but 15 conclude that the organ is probably non-functional. Contravening results which suggest that the VNO is a functional chemosensory receptor are reported by Stensaas, L., et al.. supra, and by Moran, D.T., et al . Garcia-Velasco, J. and M. Mondragon, Monti-20 Bloch, L. and B. Grosser all in J Steroid Biochem and Molec Biol. (1991) 39.

It is apparent that it would be desjrable to identify and synthesize human semiochemicals and pheromones and to develop pharmaceutical compositions 25 and methods of use to influence hypothalamic function. This invention relates to the unexpected discovery that, when nasally administered to human subjects, certain neurochemical ligands, particularly Androstane steroids, more particularly Androstene 3 0 steroids and related compounds, or pharmaceutical compositions containing Androstanes, Androstenes or related compounds, specifically bind to chemoreceptors of certain nasal neuroepithelial cells and this bind:ng generates a series of 35 neurophysiolog cal responses resulting m an hi*t , IP, _ "5" < . '1 - p *J V. 1 ' :' - , U alteration of hypothalamic function of an individual.

When properly administered, the effect of certain of these compounds on the hypothalamus affects the function of the autonomic nervous system and a 5 variety of behavioral-or physiological phenomena w'hich include, but are not limited to the following. anxiety, premenstrual stress, fear, aggression, hunger, blood pressure, and other behavioral and physiological functions normally regulated by the 10 hypothalamus Otto Appenzeller. The Autonomic Nervous System An introduction of basic and clinical concepts (1990), Korner, P.I Central nervous control of autonomic cardiovascular function, and Levy, N.M and Martin, P.J Neural control of the 15 heart, both m Handbook of Physiology; Section 2: Cardiovascular System - the heart, Vol I, Washington DC, 1979, American Physiological Society; Fishman, A.P , et al. editors, Handbook of Physiology.

Section 3. Respiratory System Vol. IX Control of 20 breathing Bethesda MD. 1986. American Physiological Society.

In some instances a single Androstane steroid, or related compound, is administered, in some instances combinations of Androstane steroids 25 and/or related compounds are administered and m some instances one or more Androstane steroids are coadministered along with one or more Estrane or Estrene steroids or a related compound.

The reader's attention is also directed to our related New Zealand Patent Specification No 257218, which describes and claims androstane steroid containing pharmaceutical compositions which are suitable for nasal administration and androstane steroid compounds all as set forth m that specification SUMMARY OF THE INVENTION Accordingly, it is an object of this invention to provide the use of ligands for a chemoreceptor displayed on the surface of nasal neuroepithelial cell m the preparation of a medicament for altering hypothalamic or autonomic function in an individual, or at least tp^ggg^d^the ^public with a useful choice OF/iz 3 o JUL 1999 I The medicament may be used to alter hypothalamic function of an individual and/or to affect physiological and benavioral functions of individuals which are normally regulated by the hypothalamus following advantages 1) administration directly to the chemoreceptors m the nasal passage and the vomeronasal organ, without pills or needles - i.e., non-mvasively, 2) a mode of drug action through the nervous system and not through the circulatory system - thus brain function can be affected without consideration of the blood-brain barrier, 3) a direct means of affecting the hypothalamus - there is only one synaptic junction between pheromone receptors and the hypothalamus; and, 4) providing a highly specific drug effect, thereby greatly reducing the potential for undesirable side-effects - this because sensory nerves are addressed to a specific location m the brain. features of the invention will be set forth m part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention invention provides a use, in the preparation of a medicament for altering a hypothalamic function of an individual, of a ligand for a chemoreceptor displayed on the surface of nasal neuroepithelial cell of said individual wherein said cell is a part of tissue other than olfactory epithelia In a further aspect, the present invention provides a use, m the preparation of a medicament for altering an autonomic function of an individual, of a ligand for a chemoreceptor of a nasal neuroepithelial cell of said individual wnerem said cell is a part .tissue other than Altering hypothalamic function may have the Additional objects, advantages and novel Accordingly, in a first aspect, the present olfactory epithelia inteucctual property ofncel RECEI i/Fn j 0 JUL 1999 OF NZ wherein P, is selected from the group consisting of oxo, a-((3~) hydroxy, a-(/3-) acetoxy, a-(j3-) propionoxy, methoxy, a-(p'-) lower acyloxy, a- (£-) lower alkyloxy, and a-(/3-) benzoyloxy; P2 is selected from the group consisting of hydroxymethyl, acyloxymethyl, alkoxymethyl, lower alkyl, hydroxyalkyl, acyloxyalkyl, and alkoxylalkyl; P3 is absent or is selected from the group consisting of hydroxymethyl, acyloxymethyl, alkoxymethyl, lower alkyl, hydroxyalkyl, acyloxyalkyl, and alkoxylalkyl, P4 is selected from the group consisting of hydrogen, oxo, halo, hydroxy, alkoxy, and acyloxy; P5 represents one or 2 substituents, wherein Ps comprises one or two hydrogen atoms, methyl, methylene, or one or two halo atoms; P6 is hydrogen or halo; and "a", "b", "c", "d", "e", "f", and "h" are alternative sites for optional double bonds.

One class of preferred steroids has "b" as t a double bond, particularly wherein "e" or "d" is also a double bond. Another preferred class has "a" and "c" as double bonds. Yet another preferred class contains P3 as a methyl group, "h" as an optional double bond, and P5 as methylene or one or two INTELLECTUAL pf.U'ERTY OrfiCE ' Or i'Z | 3 0 JUL 1999 | i,M p/" -8- \ >' ' 1 ? J J w hydrogen atoms A class of steroids wherein "a" or "b" is a double bond is also preferred By halo, it is meant, F, CI, Br, or I The term lower alkyl, lower alkoxy, etc., is meant to 5 encompass carbon chains of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms.

As noted above, altering hypothalamic function and/or autonomic function in an individual involves the use of a ligand for a chemoreceptor displaced on the surface of a nasal neuroepithelial cell The cell is a part of tissue other than olfactory epithelia, and, thQ ligand is administered within a nasal passage of the individual 15 such that the ligand binds specifically to the chemoreceptor, resulting m an alteration of hypothalamic function of the individual All embodiments of this application relate to and include the functional equivalents of the 20 steroid structures disclosed m these embodiments and to those modified steroids which demonstrate said functional equivalence, whether or not the modified steroids are explicitly disclosed BRIEF DESCRIPTION OF THE DRAWINGS 25 Figure 1 illustrates the synthesis of Androsta-4 ,16-dien-3-one, Androsta-4 ,16-dien-3cr-ol, and Androsta-4 ,16-dien-3/3-ol.

Figure 2 illustrates the synthesis of Androsta-5 ,16-dien-3a-ol and Androsta-5 , 16-dien-3j8-30 ol Figure 3 illustrates an alternate synthesis of Androsta-4,16-dien-3-one Figure 4 is a grapnic representation of "the electrophysiological effect on receptor potential of ■7 rlirruT7" L ri'fv u, f ~ | n, >7 3 0 JUL 1939 RECEIVED w'""i WI— m anrrrfWi dUmn m ,i tfjtll the localized administration of particular steroids to the vomeronasal organ of female subjects (4A) and to the olfactory epithelium (4C). Figure 4B is a graphic comparison of the effect of an Androstane on 5 the VNO receptor potential of male and female subjects Figure 5 is a graphic representation of the electrophysiological effect of the localized administration of particular steroids to the 10 vomeronasal organ of male (5A) and female (5B) subjects.

Figure 6 depicts various autonomic responses of female subjects to an Androstane. A = receptor potential of the vomeronasal 15 neuroepithelium, B = change in cortical alpha activity of an electroencephalogram (%); C = change m galvanic skin response (K-ohms); D = change m peripheral arterial pulse (counts/mm.) ; E = change in skin temperature (degrees C.); and, F = change m 2 0 respiratory frequency (counts/mm.).

Figure 7 depicts changes in receptor potential of the VNO after exposure of 5 females to two different Androstanes.

Figure 8 depicts sexual dimorphism in local 25 and autonomic responses to the stimulation of the VNO with vomerophenns. Various vomeropherins (2 00 fmoles) and the diluent control were administered to 3 0 male and 3 0 female subjects (ages 2 0 to 45 ) as described. Bars indicate the mean response of the 30 population.

Figs. 8A & B EVG responses were measured as described in male (A) and feiu^le (B) subjects.

- Figs. 8C & D- Electrodermal activity was measured as described. Changes (measured in xfl) in response due to delivery of vomeropherins to the VNO of each subject are shovm in male (C) and female (D) 5 subjects.

Figs. 8E & F: Alpha-cortical activity was measured as described. Changes m response due to delivery of vomeropherins to the VNO of male (E) and female (F) subjects.

Figs. 8G & H: Skin temperature (ST) wad measured as described. Changes m response due to delivery of vomeropherins to the VNO of each subject are shown in male (G) and female (H) subjects.

The Compounds in the graphs are: A = 1, 3, 5(10),16-Estratetraen-3-yl acetate B = Androsta-4,l6-dien-3-one C = l,3,5(10),16-Estratetraen-3-ol D = 3-Methoxy-Estra-l,3,5(10),16-tetraene E = Androsta-4,16-dien-3a-ol 20 F = Androsta- 4 ,16-dien-3/S-ol Figure 9 depicts electro-olfactgrams of male and female subjects induced by stimulation of the OE with olfactants and vomeropherins A: 400 fmoles of the olfactants 1-carvone and cineole as 25 well as 200 fmoles of the vomeropherins A, B, C, D and F; and th-2 stereoisomer E were applied separately as one second pulses to the OE of 20 subjects (both male and female) and each EOG response was recorded as described. The olfactants as well as E and B 30 produced significant (p<0.01) local response. B: 400 fmoles of the olfactants 1-carvone and cineole do not lnduce a significant EVG response when delivered to the VNO of male and female subjects.

Figure 10 depicts the electrophysiological effect of the following vomeropherins on the 5 vomeronasal organ of 2 0 female subjects: G = Androst-4-en-3-one H = Androsta-4,16-diene-3,6-dione J = 10,17-Dimethylgona-4,13(17)-dien-3-one K = 1,3,5(10),l6-Estratetraen-3-ol-methyl ether 10 L = l,3,5(10) , l6-Estratetraen-3-yl-propionate EVG = Electro-vomeronasogram GSR = Galvanic Skin Response = Electrodermal Activity, EDA ST = Skin Temperature Figure 11 depicts the electrophysiological effect of vomeropherins on the vomeronasal organ of 2 0 male subjects M = 1, 3,5(10)-Estratrien-3-ol DETAILED DESCRIPTION OF THE INVENTION 2 0 I Definitions An "affect" is a transient feeling state. Typical negative affects are feelings of nervousness, tenseness, shame, anxiousness, irritability, anger, rage, and the like. "Hoods" are longer lasting 25 feeling states such as guilt, sadness, hopelessness, worthlessness, remorsefulness, misery, unhappmess and the like. "Character traits" are more permanent aspects of an individual's personality. Typical negative character traits are sensitivity, 3 0 regretfulness, blameworthiness, stubbornness, resentfulness, bitterness, timidness, laziness and the like.

"Androstane steroids" are aliphatic polycyclic hydrocarbons characterized by a four-ring 5 steroidal structure with a methylation at the 10- and 13-positions. An Androstene is a subset of Androstanes commonly understood to mean that the compound has at least one double bond. Commonly, unless a compound is described as a gonane, it is 10 understood that the compound has a 19- carbon group. Furthermore, all derivatives which have the structural characteristics described above are also referred to generically as Androstane steroids.

A "chemoreceptor" is a receptor molecule 15 displayed on the surface of a "chemosensory" neuroepithelial cell which binds m a stereospecific fashion to a particular ligand or ligands. This specific binding initiates a signal transduction which initiates an afferent nerve impulse. 2 0 Chemoreceptors are found, inter alia, m taste buds, olfactory epithelium and vomeronasal tissue.

"Estrene steroids", as the term is used herein, are aliphatic polycyclic hydrocarbons with a four-ring steroidal structure, at least one double 25 bond m the A-rmg, no methylation at the 10-position and an oxo, hydroxy1 or hydroxy1 derivative such as an alkoxy, ester, benzoate, cypionate, sulfate or glucuronide, at the 3-position. Derivatives which contain these structural characteristics are also 3 0 referred to generically as Estrene steroids.

The following structure shows the four-ring steroidal structure common to Androstane and Estrene steroids. In describing the location of groups and substituents, the following numbering system will be employed "Sexually dimorphic" refers to a difference in the effect of, or response to, a pharmaceutical 5 agent between males and females of the same species.

An "effective amount" of a drug is a range of quantity and/or concentration which brings about a desired physiological and/or psychological effect when administered to an individual in need of the 10 drug. In the present case, a needy individual is one with a physiological or behavioral trait which is normally regulated by the hypothalamus and wherein it is desirable to affect the function of the hypothalamus or the trait. The effective amount of a 15 given drug may vary depending upon the function to be affected, the desired effect, route of administration, and the like For example, when the steroid is administered as a solution applied to the facial skin of a subject an effective concentration 20 is from 1 microgram/ml to 100 ^g/ml, preferably 10 to 50 /xg/ml and most preferably 20 to 3 0 fig/ml When the steroid is introduced directly into the VNO an effective amount is about 1 picogram to about 1 nanogram, more preferably about 10 picograms to about 25 50 picograms When the steroid is administered to the nasal passage, by ointment, cream or aerosol, or the like, an effective amount is about 100 pg to about 100 micrograms, preferably about l ng to about micrograms It follows that some drugs may be effective when administered by some routes, but not effective when administered by other routes The "hypothalamus" is the portion of the 5 diencephalon comprising the ventral wall of the third ventricle below the hypothalamic sulcus and including structures forming the ventricle floor, including the optic chiasma, tuber cinereum, mfundibulum, and mammillary bodies. The hypothalamus regulates the 10 autonomic nervous system and controls several physiological and behavioral functions such as the so-called fight and flight responses, sexual motivation, water balance, sugar and fat metabolism, hunger, regulation of body temperature, endocrine 15 secretions, and others. The hypothalamus is also the source of vasopressin which regulates blood pressure, and oxytocin which induces parturition and milk release. All hypothalamic functions are potentially modulatable by the semiochemical therapy described 2 0 herein.

A "ligand", as used herein, is a molecule which acts as a chemical signal by specifically binding to a receptor molecule displayed on the surface of a receptor cell, thereby initiating a 25 signal transduction across the cell surface. Binding of ligands to chemosensory receptors can be measured. Chemosensory tissue, such as vomeronasal neuroepithelium or olfactory neuroepithelium, contains a multiplicity of neuroreceptors cells, each 3 0 displaying at least one cell surface receptor Many of the receptor molecules have identical ligand specificity. Therefore, when the tissue is exposed to a ligand for which it has specificity (for example a exposure of the VNO to a semiochemical) a summated 7 V <1 C \ * -15- iv,' v change in cell surface receptor potential can be measured As used herein, "lower alkyl" means a branched or unbranched saturated hydrocarbon chain of 5 1 to 4 carbons, such as, for example, methyl, ethyl, n-propyl, i-butyl and the like. "Alkoxy" as used herein is used m its conventional sense to mean the group -OR wherein R is alkyl as herein defined.

A "pheromone" is a substance that provides 10 chemical means of communication between members of the same species through secretion and nasus reception. In mammals pheromones are usually detected by receptors m the vomeronasal organ of the nose Commonly, pheromones effect development, 15 reproduction and related behaviors. A "semiochemical" is a more general term which includes pheromones and describes a substance from any source which functions as a chemosensory messenger, binds to a specific neuroepithelial receptor, and induces a 20 physiological or behavioral effect. A "vomeropherm" is a semiochemical whose physiologic effect is mediated through the vomeronasal organ A picogram (pg) is equal to 001 nanograms (ng) . A ng is equal to .001 micrograms {jxg) . A /ig 25 is equal to .001 mg.

II. Modes for Carrying Out the Invention A. Androstanes useful in the Invention The invention makes use of a group of certain Androstane steroids. Testosterone (17-30 hydroxy-Androsta-4-en-3-one) is a typical Androstane.

Androstanes especially suitable for use m the present invention include those where, independently, pt = oxo, cr-hydroxy, /S-hydroxy; P2 = £ V imellcctijm ?iii erftumc:i r~ [ 1 2 3 0 JUL 1999 RECEIVED V methyl/ lower alkyl, hydroxymethy1, hydroxyalkyl; P3 = hydrogen or methyl, P4 = hydrogen, hydroxy, or oxo; P5 = hydrogen or methyl; and there is at least one double bond, asually at the 4- or 16- position.

Preferred Androstanes include Androsta- 4, i6-dien-3-one (P, = oxo, a = double bond, P3 = methyl, P3, P4, P5 = hydrogen, commercially available from Steraloids, Inc.), Androsta-4,16-dien-3/?-ol (P, = £-0H, a = double bond, P2 = methyl, P3, P4/ P5 = 10 hydrogen), and 6-keto-Androsta-4,l6-diene-3-one (P, = oxo, a = double bond, P: = methyl, P3, P5 = hydrogen, P4 = oxo), synthesis of which are described herein.

A subset of Androstanes within the group are believed to be novel. Syntheses are described 15 herein for the following compounds as designated on the chart 17-methylene-Androst-4-en-3B-ol (A3/N3), 17-methylene-Androst-4-en-3a-ol (A4/N3), 17-methylene-6-oxo-Androst-4-en-3-one (A6/N3), and 6B-OH-Androsta-4,16-dien-3-one (All/Nl). 20 Chart 1 includes androstanes which are useful m the invention, but do not limit its scope The synthesis diagrams that follow depict intermediate and substructure syntheses for the preparation of these androstanes.

M rELLECT UAlTFI - ERTY 0?r, oE~ 1-" i JI i 0 JUL 1993 RECEIVED «n~» A * CHART I ANDROSTANES /KN 1 2 3 4 1 &5° V KNOWN 0X^° ° KNOWN U KNOWN 0J35^ ° KNOWN 2 Hfl KNOWN "0^N HO-^ KNOWN £3$^ HO^^ 3 "0-'C^N .C653 H0' KNOWN 4 x£° HO KNOWN HO^n HO"13515 c/^S13 KNOWN JSS0 u KNOWN pS6 S °KNOWN 0^ U KNOWN 0&96 0 7 O-St?3 KNOWN oS?3 £5* 8 M6° KNOWN uo&$° MGO KNOWN ..0^ 9 oJSt?3 u KNOWN ° KNOWN 0d5l} HO^ HO-15?5 HO^^ 11 0^ OH OHKNOWN OH flwiEU -*r f< 7 Wl 1 3 0 JUL 1999 > r- cu/pn « flUBSTRUCTURE SYNTHESES Referring to the preceding table, the following are exemplary syntheses for intermediates in a given row (Al through All) or column (N1 through N4) Type A Al o^b O noxb Q. Odo (A2) (A1) HO O ^V A2: xb This is a commercially available substructure, for example, DEHYDRO EPI ANDROSTERONE. 9 ^3 xb HO NaBH4 • xb HO (A1) (A3) NaBH4 60% (Michio Matsui and David K Fukushima, J Org 1970, Vol 35, No 3, p 561-564) A4 HO .db xb Li[CH(CH 2)CH(CH3)2|3BH ► HO (Al) ^jcxy u[ch(ch 3)ch(ch3) 48% 2I3BH t Ohloff, C- ec al fHelv Chim Acta (1983) 66 192-217) o o do CHLORANIL • 0£b (Al) CHLORANIL 45% German Off 2,631,915 AG HO O xb Cr03/H25Q4 (A2) wXi) HO CrO^HgSO^ 36% ' (A5) (A6) J Rcmer, H Wagner, and W Sihade, Steroids, 1988, 51/5-6, p 577-581) CH-OH .do CHjOH <toxb —odo (Acetate of A2) (A7) §2!!se^ r|^ AcO (Hacermehl, et al , Z Naturforsch (1980) 256 191-195) A8 jdo MeO rHk> m*oxom*/h+1i Xt) MeO (Al) (A8) ""QXQ^/H4, 44% MeO A5 xb (A1) DICHLORODICYANOBENZOQUINONE - (a9) DICHLORODICYANOBENZOQUINONE 43% Ohloff, G et al 192-217) A10 xb HO (Helv Chim Acta (1983) £6 ,j±I (A9) LiAIH HO Xb (A10) O LiAIH 30% HO V I Mel'nikova and K K Pivnitskii, Zhurnal Organickeskoi Khisnn, 1972, Vol 8, No 1, pp 68-74) 9 All OH MeO jC£) (A3) MeO -SEE EXAMPLE 19— ■23- MCPBA (AH) OH MCPBA - O i OH * Tvne N N1 b 0 N-NKTs TsNHNH, fA n 3uLi ^ TsNHNH,( ^ HO TsNHNH 95% HO b (Nl) N-NHTs n-BuLi 50% HO N2 b ^ n2h/<oh [ (n2) HO HO H</KOH HO HO 1 Robert K Shapiro and Carl Djerassi, J Am Chem Soc , 1964, 86, 2825 2 Pilar Lupon, Frances C Canals, Arsenio Iglesias, Joan C Ferrer, Albert Palomar, and Juan-Julio Bonec, J Org Chem 1908, 53., 2193-2198 -25 N3 as in & HO I/V PhaP=PI; & Ph.,p=qH; 64% b (N3) HO and joSP* ,P=CH L, 50% HO HO 1 Guncher Drefahl, Kurt Ponold and Hans Schick, Benchte, 1965, jJJ., 604 2 Richard H Peters, David F Crows, Mitchell A.

Avery, Wesley K M Chong, and Masako Tanabe, J Med. Chem , 1989, 12, 1642 N4 6 u in OTi 6 COLUDINE OTs COLUDINE 6 (N4) ind OH W"° MeO 0 1 Franz Sondheimer, O Mancera, M Urquiza & G Rosenkranz, J Am Chem Soc , 1955, 77, 4145 William F Johns, J Org Chem , 1961, 26, 4583 Metnvlandrostenes German Off 2,631,915 teaches preparation of 0 with a methyl group at any one of the following positions la, 2a, 4, 6a, 6£, 7a, and 16 Daniel Bertm and Lucien Nedelac, Memoires Presentes a la Societe Chimique, 1964, No 345, p 2140 Syntnesizable compounds therefore include these, together with those derived from them, i e , N1 with methyl at la, 2a, 4, 6a, 66, 7a, 16 or 17 combined wich Al, A3, A4, A5, A8, A9, A10 or All, as well as A2 or A6 with a 17-methyl O 6-METHYLANDROSTA-4.6-DIEN-3-ONE German Off 2,428,679 Synthesis of the 17-M ETHYLANDROSTENES Haloandrostenes U.S. Patent 3,413,321. a HO ^ j.

HO a o j o Br I a o J o i o oj^5b 0^Ji> a a a a Br German Off. 2,631,915.

V- V- \ ' 1 .

Br HO European Pacer.: Application E? 208,497 Syntnesizaole compounds tnerefore include these, togetner witn tnose derived from them, i e , (4-Chlorc, 4-5romo, Sa-Chloro, 6a-Bromo, 66-Chloro, 66-3romo, or Sfi-Icao^-Al ir commation witn N1, N2, N3, or N4 Ii adaption, (17-Fluoro, 17-Chloro, 17-3romo, or 17-Iodo)-Nl m combination with Al, A2, A3, A4, A5, AS, A3, A9, A10 or All The invention is additionally directed to compositions of New Zealand Patent Specification No 257218 involving the combination of tne aforementioned Androstane steroids witn certain Estrene steroids wmcn are structurally related to Estradiol (also referred to as 1, 3 , 5 (10)-Estratnene-3,173-diol) Estrene steroids useful m this invention have the formula B Estrenes useful m the Invention R R6 ^4 !i\ilElLCPTIlAL PRCt LRTV OFFIC '> i Z 3 0 JUL 1999 RECEIVED whcrem R,is selcctod from the group consisting essentially of one or two hydrogen atoms, methyl, methylene, and one or two halo atoms; R, is absent or is selected from the group consisting essentially of 5 hydrogen and methyl; R3 is selected from the group consisting essentially of oxo, hydroxy, lower alkoxy, lower acyloxy, benzoyl, cypionyl, glucuronide and sulfonyl, R4 is selected from the group consisting essentially of hydrogen, hydroxy, lower alkoxy, lower 10 acyloxy and halo; R5 is absent or is selected from the group consisting essentially of hydrogen, hydroxy, lower alkoxy and lower acyloxy; is a hydrogen or a halo; and "a" represents optional aromatic unsaturation of ring A of said steroid, or 15 "b", "c", and "d" are each optional double bonds; and "e", "f", "g", "h", "1" and "3" are each optional double bonds. In this embodiment, the steroid is preferrably administered m the form of a pharmaceutical composition containing one or more 20 pharmaceutical^ acceptable carriers.

A preferred class of compounds are those m which "a" is present and "g", "h" or "1" are optional double bonds The class wherein "h" and "iM are both double bonds is also preferred. Another preferred 25 class contains "b", "c" or "3" as a double bond Yet another class contains "c" and "d" as double bonds. Still another class contains R2 as methyl and (1) "e" as a double bond, (2) R, is methylene or a single hydrogen, or (3) "f" is a double bond. 30 Preferred estrenes include 1,3,5(10)- Estratriene-3 , 17/3-diol; 1, 3 , 5 (10)-Estratriene-3 ,16a, 17/S-triol; 1,3,5(10) -Estratriene-3-ol-17-one; 1,3,5 (10),l6-Estratetraen-3-ol; 1,3,5 (10),16-Estratetraen-3-ol methyl ether, and 1,3,5(10),16-35 Estratetraen-3-yl acetate.

Cj / Most, of these steroids and their glucuronide, sulfate, cypionate, and benzoate derivatives, are compounds known m the art and are commercially available, e.g., from Sigma Chemical 5 Co., Aldrich Chemical Co., etc. Alkoxy derivatives and their syntheses are also known in the art and shown m U.S. Patent No. 2,984,677. 1,3,5(10),16-Estratetraen-3-ol is available from Research Plus, Inc. and from Steraloids, Inc.

Another synthesis of this compound, as well as syntheses of the acetate and propionate derivatives are described in our commonly owned New Zealand Patent Specification No 256796 C Synthetic Methods 1. Preparation of 3-, 5-, 6-, 18-and 19- position derivatives.

The compounds used m this 20 invention are Androstane steroids substituted at the 3-, 5-, 6-, 18- and 19- positions Many of the 3-and 5-substituted steroids are known compounds which may be derived from 17-hydroxy-and 17-oxo-steroids (commercially available e g. from Aldrich Chemical 25 Co) by elimination or reduction to the ^16 homologue. The syntheses of most of these compounds are described by Ohloff (supra). As shown in Figure 1, 17/3-hydroxy-5a-A_ndrostan-3-one (I) and methyl chlorofornate (a) in pyridine gives the methyl 30 carbonate, 17/3-raethoxycarbonyloxy-5a-Androstan-3-one (II) which provides a starting material for the 5a-Androst-16-en-(3-one and 3-ols) (Ohloff, supra at pg 200) INTELLECTUALVfiQJERTi OrFiCf IJZ 3 0 JUL 1939 RECEIVED This compound has been disclosed as an intermediate in the synthesis of 19-oxo-3-aza-A-homo-5B-androstane (Habermehl, et al f z Naturforsch (1970) 25b.191-195). A method of synthesizing this 5 compound is provided D Pharmaceutical Compositions and Methods of Use Described but not claimed is a method of altering the hypothalamic function of an individual.

Also described is a method of altering an autonomic function of an individual. These autonomic functions include but are not limited to heart rate, respiratory rate, brain wave patterns (percentage alpha cortical activity), body temperature. Other methods described include, but are not limited to, methods of diminishing negative affect, negative mood or negative character traits of an individual. Another method described is of treating female premenstrual stress All of these methods are 2 0 accomplished by means of the non-systemic, nasal administration of certain 16-Androstene steroids, combinations of 16-Androstene steroids and combinations of one or more 16-Androstene steroids and one or more Estrene steroids.

This particular mode of administration is distinguished from alternative modes, such as ingestion or infection, in several important ways, these by virtue of the direct contact with the VNO provided by the nasal administration of the steroid 3 0 ligand. In the methods described, the appropriate ligand is administered directly to the chemoreceptors in the nasal passage and the vomeronasal organ, without pills or needles - i.e., non-invasively Drug action is mediated through INTELLECTUAL PROPERTY 0FT((,E"i OF tiZ $ 0 JUL 1999 Received This compound has been disclosed as an intermediate in the synthesis of 19-oxo-3-aza-A-homo-5B-androstane (Habermehl, et al . Z Naturforsch. (1970) 25b.191-195)■ A method of synthesizing this 5 compound is provided.

D Pharmaceutical Compositions and Methods of Use An embodiment of the subject invention is a method of altering the hypothalamic function of an 10 individual. Another embodiment is altering an autonomic function of an individual. These autonomic functions include but are not limited to heart rate, respiratory rate, brain wave patterns (percentage alpha cortical activity), body temperature. Other 15 embodiments include, but are not limited to, methods of diminishing negative affect, negative mood or negative character traits of an individual. Another embodiment is a method of treating female premenstrual stress. All of these embodiments are 20 accomplished by means of the non-systemic, nasal administration of certain 16-Androstene steroids, combinations of 16-Androstene steroids and combinations of one or more 16-Androstene steroids and one or more Estrene steroids.

This particular mode of administration is distinguished from alternative modes, such as ingestion or injection, m several important ways, these by virtue of the direct contact with the VNO provided by the nasal administration of the steroid 30 ligand. In the methods of this invention, the appropriate ligand is administered directly to the chemoreceptors m the nasal passage and the vomeronasal organ, without pills or needles - i.e., non-invasively Drug action is mediated through bmding of the ligands, described herein, to specific receptors displayed by neuroepithelial cells in the nose, preferably in the VNO. This Furthermore, the mode of drug action is through the nervous system and 5 not through the circulatory system - thus brain function can be affected without consideration of the blood-brain barrier. These methods of treatment provide a direct means of affecting the hypothalamus through the nervous system because there is only one 10 synaptic junction between pheromone receptors and the hypothalamus. Because sensory nerves are addressed to a specific location in the brain, this method has a highly specific drug effect, thereby greatly reducing the potential of undesirable side-effects. 15 VNO contact is important because the VNO is associated with chemoreceptive/pheromonal function. The VNO consists of a pair of blind tubular diverticula which are found at the inferior margin of the nasal septum. The VNO contains neuro-epithelia, 2 0 the axons of which have direct synapses to the amygdala and from there, to the hypothalamus. The existence of the VNO has bean well documented in most terrestrial vertebrates including the human fetus/ however, in adult humans it is generally thought to 25 be rudimentary (See Johnson, et al.. supra).

The ligand substances described herein, or their sulfated, cypionated, benzoated, propionated, or glucuronated derivatives, may be administered directly, but are preferably administered as 3 0 compositions. They are prepared in a liquid dosage form such as, for example, liquids, suspensions or the like, preferably in unit dosage forms suitable for single administration of precise dosages. Liquid dosages may be administered as nose drops or as an 35 aerosol. Alternatively, the active compound can be -3 4- prepared as a creme or an ointment composition and applied topically within the nasal cavity As another alternative, delivery may occur by controlled release of these agents by encapsulation either in 5 bulk or at a microscopic level using synthetic polymers, such as silicone, and natural polymers such as gelatin and cellulose. The release rate can be controlled by proper choice of the polymeric system used to control the diffusion rate (Langer, R.S. and 10 Peppas, N.A., Biomaterials 2,201, 1981). Natural polymers, such as gelatin and cellulose slowly dissolve in a matter of minutes to hours while silicone remains intact for a period of months. The compositions will include a conventional 15 pharmaceutical carrier or excipient, one or more of the active 16-Androstene compound(s) of Formula I, and the composition may or may not additionally include one or more Estrene steroids. In addition, the compositions may include other medicinal agents, 2 0 pharmaceutical agents, carriers, adjuvants, etc.

The most likely means of communication of a semiochemical ligand is the inhalation of a naturally occurring pheromone present on the skm of another. Several 16-Androstene steroids, including 5a-Androst-25 16-en-3a-ol and 5a-Androst-16-en-3-one, 4, 16-Androstadien-3-one, 5a-Androstadien-3/3-ol, and perhaps 5a-Androstadien-3a-ol, are naturally occurring m humans and may be present on the skin. It is estimated that the naturally occurring maximum 3 0 concentration of a 16-Androstene steroid on human skm is from 2 to 7 ng/cm2. During intimate contact it is estimated that a human would be exposed to no more than 700 ng of a naturally occurring steroid. Since these compounds are relatively nonvolatile, it 35 is estimated that, even during intimate contact, a human subject would inhale no more than 0.7 pg of a naturally occurring steroid from the skin of another. From the amount inhaled only about 1% would reach the receptors of the vomeronasal organ. Thus the 5 estimated maximum natural exposure to naturally produced pheromones would be 0.007 pg.

The amount of semiochemical ligand administered will of course, be dependent on the subject being treated, the severity of the 10 affliction, the manner of administration, the frequency of administration, and the judgment of the prescribing physician However, a single dosage of at least about 10 picograms, delivered directly into the lumen of the vomeronasal organ, is effective m 15 eliciting a transient autonomic response. When administered to the nasal cavity, the dosage is about 10 0 picograms to about 100 micrograms, preferably about 1 nanogram to about 10 micrograms, more preferably about 10 nanograms to 1 about microgram. 20 The frequency of administration is desirably m the range of an hourly dose to a monthly dose, preferably from 8 times/day to once every other day, more preferably 1 to 3 times per day. Ointments containing one or more active compounds and optional 25 pharmaceutical adjuvants m a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, can be prepared using a base such as, for example, petroleum jelly, lard, or lanolin. 3 0 Liquified pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, 3 5 aqueous dextrose, glycerol, ethanol, and the like, to -3 6- thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH 5 buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, 10 see Remington's Pharmaceutical Sciences. Mack Publishing Co., Easton, PA, 15th Ed., 1975 The composition or formulation to be administered will, in any event, contain a quantity of one or more of the active compound(s) in an amount effective to 15 alleviate the symptoms of the subject being treated.

For aerosol administration, the active ingredient is preferably supplied m finely divided form along with a surfactant and a propellant.

Typical percentages of active ingredients are 0.001 20 to 2% by weight, preferably 0.004 to 0.10%.

Surfactants must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 2 5 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, lmoleic, olestearic and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride such as, for example, ethylene glycol, glycerol, erythritol, arabitol, mannitol, 3 0 sorbitol, and hexitol anhydrides derived from sorbitol (the sorbitan esters sold under the trademark "Spans") and the polyoxyethylene and polyoxypropylene derivatives of these esters. Mixed esters, such as mixed or natural glycendes, may be 35 employed The preferred surface-active agents are the oleates or sorbitan, e.g , those sold under the trademarks "Arlacel C" (sorbitan sesquioloate), "Span 80" (sorbitan monoleate) and "Span 85" (sorbitan trioleate). The surfactant may constitute 0 1-20% by 5 weight of the composition, preferably 0 25-5%.

The balance of the composition is ordinarily propellant Liquefied propellants are typically gases at ambient conditions, and are condensed under pressure. Among suitable liquefied 10 propellants are the lower alkanes containing up to five carbons, such as butane and propane; fluormated or fluorochlorinated alkanes, such as are sold under the trademark "Freon" Mixtures of the above may also be employed.

In producing the aerosol, a container equipped with a suitable valve is filled with the appropriate propellant, containing the finely divided active ingredient and surfactant. The ingredients are thus maintained at an elevated pressure until 20 released by action of the valve.

Yet another means of administration is topical application of a volatile liquid composition to the skm, preferably facial skin, of an individual. The composition will usually contain an 25 alcohol such as ethanol or isopropanol A pleasant odorant may also be included in the composition.

F Measuring Affect. Mood and Character Trait.

Feeling states associated with affects, 3 0 moods and character traits are generally measured by use of a questionnaire. For example questionnaires comprising a number of adjectives which refer to feeling states may be administered to an individual. The individual evaluates his or her feeling state described by the adjective and rates the intensity of the feeling on a numerical scale. Clustering of re lated adjectives and statistical analysis of a subject's evaluation of each adjective provides a 5 basis for the measurement of various feeling states measured by autonomic changes, such as those used in polygraphic evaluations (galvanic skin response, pulse rate and the like). Cabanac, M. Annual Review 10 of Physiology (1975) 17:415, Hardy, J.D., "Body Temperature Regulation", Chapter 59, pp. 1417 In: Medical Physiology. Vol. IIEd : VB Mountcastle (1980); Wolfram Bouscein Electrodermal Activity (Plenum Press 1992) In addition, non-verbal cues 15 such as facial expression and body posture may be evaluated. 2 0 intranasal administration, and which contain one or more of the steroidal substances described herein, are useful as therapeutics with efficacy m the treatment of certain types of psychiatric disorders, particularly certain types of neuroses. In a 25 preferred embodiment, and unlike other drug formulations designed to invoke a systemic effect, the composition is formulated to minimize the likelihood of mucosal or transdermal absorption since the mode of action of these substances as 3 0 therapeutics is by stimulation of neural receptors m the nose, more particularly in the VNO. Since the compositions useful m the instant invention are effective as the result of stimulation of chemosensory receptors m the VNO and not as a result of systemic 3 5 circulation, absorption and the attendant Alternatively, feeling states may be G Uses in the Treatment of Certain Types of Psychiatric Disorders Compositions suitable for topical and/or 10 39- mcorporation of the active ingredient or ingredients into systemic circulation would not be efficacious, but rather might actually increase the likelihood of side effects These compositions are useful when 5 administered in a method such that one or more of the Estrenes or 16-Androstenes of the composition are provided to the VNO of the subject. transient or they may be persistent or recurrent.

They generally involve abnormal symptoms that may include mood changes (anxiety, panic, depression) or limited abnormalities of thought (obsessions, irrational fears) or of behavior (rituals or compulsions, pseudoneurological or hysterical conversion signs). In such disorders, these compositions may have some beneficial effects for short periods, particularly by modifying associated anxiety or depression or the like. 2 0 be treatable by the intranasal administration of the compositions useful in this invention These conditions include characteristic personality styles, e.g., paranoid, withdrawn, psychopathic, hypochondrial and the like, or behavioral patterns, e.g., abuse of 25 alcohol or other substances, socially deviant or perverse behavior and the like that may run counter to societal expectations. conditions, both normal (such as menstruation) and 30 disease or injury related (such as chronic illness) may have a psychological component that manifests as a psychiatric disorder, neurotic disorder, or as an anxious or depressed affect or mood These conditions are also treatable by methods of 35 administration of the compositions Treatable neuroses may be acute and/or Other characterological disorders may also Certain physical or physiological INTELLECTUAL PRGr,ERT> OFFICE OF NZ 3 0 JUL 1999 RECEIVED mcorporation of the active ingredient or ingredients into systemic circulation would not be efficacious, but rather might actually increase the likelihood of side effects These compositions are useful when 5 administered in a method such that one or more of the Estrenes or 16-Androstenes of the composition are provided to the VNO of the subject.

Treatable neuroses may be acute and/or transient or they may be persistent or recurrent. 10 They generally involve abnormal symptoms that may include mood changes (anxiety, panic, depression) or limited abnormalities of thought (obsessions, irrational fears) or of behavior (rituals or compulsions, pseudoneurological or hysterical 15 conversion signs). In such disoiders, these compositions may have some beneficial effects for short periods, particularly by modifying associated anxiety or depression or the like.

Other characterological disorders may also 20 be treatable by the intranasal administration of the compositions of this invention. These conditions include characteristic personality styles, e.g., paranoid, withdrawn, psychopathic, hypochondrial and the like, or behavioral patterns, e.g., abuse of 25 alcohol or other substances, socially deviant or perverse behavior and the like that may run counter to societal expectations.

Certain physical or physiological conditions, both normal (such as menstruation) and 30 disease or injury related (such as chronic illness) may have a psychological component that manifests as a psychiatric disorder, neurotic disorder, or as an anxious or depressed affect or mood. These conditions are also treatable by methods of 35 administration of the compositions of the present hr/ ; t such that one or more of the estrenes or androstenes of the composition are provided to the VNO of the subject. 1 Anxiety Anxiety is a disagreeable emotional state characterized by feelings of uneasiness, impending danger, apprehension or tension Compositions suitable for intranasal administration, and which contain one or more of the Estrene steroid substances 10 described herein, are useful as therapeutics with efficacy in the reduction of anxiety. There are numerous manifestations of this disorder ranging from mild perturbations to syndromes which can be incapacitating Anxiety is not only a cardinal 15 symptom of may psychiatric disorders but also a component of many normal physiological and social conditions In addition, symptoms of anxiety are commonly associated with depression and especially with dysthymic disorder (neurotic depression) and 20 many personality disorders Anxiety disorders can be separated and include panic disorders, phobias and generalized anxiety disorders, post-traumatic stress disorder and obsessive-compulsive disorder. li Drugs used m the treatment of anxiety 25 Drug treatment is the major treatment method of anxiety, often used in conjunction with behavior therapy. The drugs used most frequently to treat anxiety are the benzodiazepines.

Benzodiazepines have hypnotic, sedative, anxiolytic, 3 0 anticonvulsant and muscle relaxant actions.

Consequently, they are used for may indications and in many different conditions than anxiety disorders (e.g , insomnia, alcohol withdrawal states, muscle spasms, epilepsy, anesthesia and sedation for 3 5 endoscopic procedures). These drugs are relatively 'iELt[f TU4l~ plto i cjTY oi~FluL * | , J o JUL 1S99 j LillPEivED I safe, and have advantages in comparison with the previously used barbiturates. They have a rapid onset of action, and produces a sense of euphoria. However, despite their good safety record, 5 benzodiazepines are associated with a number of properties that limit their use including sedation, ataxia, memory impairment, reduced motor coordination and dangerous addictive effects, especially when used with alcohol. Both physicians and patients are 10 increasingly aware of this potential for addiction; and there is an evident desire, if not trend, to move away from their longterm use.

Recently, a new class of non-benzodiazepme anxiolytic agents, azaspirodecanediones, which may 15 have more selective anxiolytic properties, has been approved. The first marketed member of this class is buspirone. This compound has been reported to be as effective as benzodiazepines in the treatment of Generalized Anxiety Disorder. Compared to 20 benzodiazepines, buspirone is less sedating, potentiates the effect of ethanol to a lesser degree, and has a low addictive potential However, m normal clinical use, it has been found to be difficult to substitute buspirone for benzodiazepines 25 in patients already receiving benzodiazepines since buspirone does not create a feeling of euphoria, and patients do not feel as satisfied as when taking benzodiazepines. In addition, buspirone's slow onset of action (1-3 weeks) limits its effectiveness m 3 0 treating acute anxiety. Systemically administered buspirone anxiolytic has been shown to have a high affinity for the CNS serotonin 5-HT receptor and this is thought to be the mode of anxiolytic action Another class of new anxiolytic is the 3 5 systemically administered benzofuran derivatives. ? *7' f'» ' \ ) _42- V These substances demonstrate a high affinity for the CNS dopamine D2 receptor 111 Mood (Affective) disorders The compositions described 5 are also useful in the treatment of some of the symptoms of certain types of mood disorders. Mood disorders such as major depression and mania are characterized by changes in mood as the primary clinical manifestation. Either extreme of mood may 10 be associated with psychosis, characterized by disordered delusional thinking and perceptions, often congruent with the predominant mood. Conversely, psychotic disorders may have associated or secondary changes in mood Similarly, normal grief, sadness 15 and disappointment and the dysphoria or demoralization often associated with medical illness or with normal cyclic dysfunctions such as premenstrual stress may also be mitigated by the intranasal application of the compositions of the 20 instant invention. iv The mode of action of the compositions of the instant invention The mode of action of the compositions useful m the instant invention is quite different from any 25 modes of treatment that have been heretofore reported. The active steroid and steroid-like compounds described stimulate and/or bind to receptors in the VNO that are directly available to topical administration. 30 Direct access to CNS receptors is not required Stimulation of these receptors then produces a signal transmitted through a neural pathway and inducing a f IN I El LECTUAL "PPC - ERTY OFFICE I n'r 1L i 3 0 JUL 1909 | _ RECEIVFn I A "*r -4 3- V} U > nGuropsychic response, likely m the hypothalamic region of the subject's brain. This response manifests as discrete changes m a variety of autonomic functions, including but not limited to 5 pulse rate, respiratory frequency, E.E.G. patterns, evoked potential, skin temperature, galvanic skin response and pupillary diameter. The response also manifests as a measurable reduction of negative affect and negative mood.

While the relationship between the modification of hypothalamic function and the treatment of psychiatric disorders is not fully understood, it is clear that the compositions are effective in the reduction of self-15 evaluative negativity - negative mood characteristics as well as negative character. These changes are accompanied by autonomic changes, effected by the hypothalamus, that are consistent with an increase in parasympathetic tone (or alternatively a decrease m 2 0 sympathetic tone).

Since the active compounds are pheromone-like in their specificity, the compounds exhibit a species specificity m their stimulation and activity add therefore demonstration 25 of efficacy cannot be done m animal models. flNraiECTUAL PR^CrtlTM J >> IU 3 0 JUL 1909 III. Examples The following examples are intended to illustrate but not to limit the invention.

Abbreviations used in the examples are as 5 follows: aq.= aqueous; RT.=room temperature; PE=petroleum ether (b.p. 50-70'), DMF=N,N-dimethylformamide; DMSO=dimethyl sulfoxide; THF=tetrahydrofuran.

Example 1 - Androsta-4 .16-dien-3-one M). 10 This synthesis is depicted in Figure l.

Several methods are known for the conversion of testosterone into Androsta-4,16-dien-3-one (Brooksbank et al . Biochem J. (1950) AZ:36). Alternatively, thermolysis (460°) of the methyl 15 carbonate of testosterone gives Androsta-4,16-dien-3 one in 9 0% yield. 17B-MethoxyCarbonyioxy-androst-4-en-3-one (iv) was prepared from testosterone (III. Fluka) with methyl chloroforraate/pyridme (a) in 76% yield (after recrystallization from MeOH). M.p. 140 20 141°, [a]D = +95.4° (c = 1.10) - IR (CDC13) : 1740s, 1665s, 1450s, 1280s, - 'H-NMR. (360 MHz): 0.87 (s, 3 H) , 1.20 (s, 3 H) ; 3.77 (s, 3 H) ; 4.53 (br. t, J 8, H); 5.75 (s, 1 H). A solution of the methyl carbonate IV in toluene was pyrolyzed (b) as 25 described for I. Recrystallization of the crude product from acetone at RT. gave pure ketone 4 in 90% yield. M.p. 127-129.5°, (a)D +118.9° (C = 1.32) ([3]: m.p. 131.5-133.5° (hexane), [a]D16 = +123 + 3.5° (C = 1.03)). - IR. (CDC13) : 3050w, 1660s, 1615m. - lH-5 NMR. (360 MHz): 0.82 (s, 3 H)7 1.22 (s, 3 H); 5.70 (m, 1 H); 5.73 (s, 1 H); 5.84 (m, 1 H).

Example 2 - Androsta-4.16-dien-3g-ol f5) and -3fl-ol 111.

These syntheses are depicted m Figure 1. 10 Androsta-4,16-dien-3-one (4) was reduced at -55° with lithium tris (1, 2-dimethylpropyl) hydridoborate in THF (c) as described for the preparation of 2 (Figure 1) . Chromatography on silica gel with CH2Cl2/ethyl acetate 9:1 gave pure axial alcohol 5 (48% yield) and 15 pure equatorial alcohol 6 (48% yield). Analytical samples were further purified by recrystallization (from PE at -30° for 5, from cyclohexane at RT. for 6) .

Data of 5. M.p. 77-79°, [a]D +120.6° (C = 20 1.26) - IR. (CDClj) : 3620m, 3440m br., 1660m, 1595W. -4i-NMR. (360 MH2): 0.79 (s, 3 H) , 1.02 (s, 3 H) ; 4.07 (m, w1/4 ss 10, 1 H) ; 5.48 (d x d, J 5 and 2, 1 H) ; 5.71 (m, 1 H) ; 5.85 (m, l H) .

Data of 6 M.p. 116.1190, [a]D +53.9° (C 25 = 1.28) ([47): m.p. 116.1180, [8)D +59.3° (C = 0.4) - IR. (CDClj) : 3610m, 3420m br., 3050m, 1660m, 1590W. - 'H- NMR. (360 MHz): 0.78 (s, 3 H); 1.08 (s, 3 H); 4.15 (m, ~ 20, 1 H) ; 5.30 (m, w,A ~ 5, l H) ; 5.71 (m, 1 H) ; 5.85 (m, 1 H).

Example 3 - Androsta-5.16-dien-3a-ol (7).

This synthesis is depicted in Figure 2. To a solution of alcohol 8 (545 ing, 2.0 mrnol) in acetone (100 ml) at 0°C under N:was added rapidly Jones reagent (l, 1.5 ml, ca. 4 mmol) . After 5 mm., the 10 mixture was poured into a dilute phosphate buffer (pH 7.2, 1200 ml) and extracted with ether. The extracts were washed with sat. aq. NaCl solution, dried (Na^SO.,) and evaporated to give mainly Androsta-5,16-dien-3-one as an oil (567 mg). The crude product was 15 dissolved In THF (7 ml) and reduced with lithium tris (1,2-dimethylpropyl) hydridoborate (c) at .55° as described for the preparation of 2. The crude product (53 0 mg) was chromatographed on silica gel (100 g) with CH2Cl2/ethyl acetate 4:1 to give 280 mg 20 (51%) of pure a-alcohol 7 (eluted first) and 13 mg of starting alcohol 8. A small sample of 7 was recrystallized from acetone/water at RT. M.p. 1380, [8 ] D -77.5° (c= 1.2. - IR (CDC13) 3580m, 3430m, 1665w, 1590w, - lH-N"MR. (360 MHz). 0.80 (s, 3 H) ; 1.06 (s, 3 H) ; 4.02 (m, w^.8, 1 H) ; 5.44 (m, 1 H) ; 5.72 (m, 1 H); 5.86 (m, 1 H).

Example 4 - Androsta-5.16-dian-3B-ol (8).

This compound was prepared in 73% yield by 5 a known procedure (Marx, A.F., et al . Ger. Offen. 2,631,915; Chem. Abst. 87:23614P (1977)) from commercial (Fluka) 3B-hydroxy-androst-5-en-17-one (VII). M.p. 137°, [a]D = -71.9° (c=1.5) ([48]: m.p. 140-141°, [a]D = 68°. - IR. (CDClj) : 3600m, 3420m 10 br., 1670W, 1590w, - 'H-NMR. (360 mh2) : 0.80 (s, 3 H) ; 1.05 (s, 3 H); 3.53 (m, ww«22, 1 H); 5.38 (m, 1 H) ; 5.72 (m, 1 H); 5.86 (m, 1 H). This synthesis is depicted m Figure 4.

Example 5 - Alternate synthesis of Androsta-4.16-15 dien-3-one (25).

The following method of synthesis is depicted in Figure 3: Dehydroepiandrosterone p-Toluenesulfonylhydrazone (23) Dehydroepiandrosterone (VII) (14.4 g, 50.0 m mole) and p-toluenesulfonylhydrazide (12.75 g, 68.5 m mole) in dry methanol (300 ml) were heated under reflux for 20 hours. The mixture was transferred to a conical flask arid allowed to cool. The crystalline product was faltered under suction and washed with methanol (50 ml). Further crops of product were obtained by sequentially evaporating the filtrate to 5 75 ml and 20 ml, and allowing crystallization each time. Total yield was 21.6 g (95%), Androsta-5, 16-dien-3/3-ol (24) Dehydroepiandrosterone p-toluenesulfonylhydrazone (23) (22.8g, 50.0 m mole) m 10 dry tetrahydrofuran (1.0 liters) was cooled in a dry ice/isopropanol bath, The-mixture was stirred while n-butyl lithium (12 5 ml of l 6 M solution in hexane, 200 m mole) was added. The mixture was allowed to warm to room temperature and was stirred for 2 4 15 hours. Water (50 ml) was added with cooling in ice. The mixture was poured into saturated ammonium chloride solution/ice (500 ml) and extracted with ether (x2). The organic layers were washed with saturated sodium bicarbonate solution (50 0 ml) and 2 0 saturated sodium chloride solution (500 ml), dried (MgS04) and evaporated in vacuo to give the crude product. This was purified by flash chromatography on 190 g silica gel 60, 230-400 mesh, elutmg with ethyl acetate/hexane (2 0:8 0—50•50) to give crystalline material. The product was recrystallized from methanol (45 ml)/3% hydrogen peroxide -(8 ml) washing with methanol (30 ml)/water (8 ml) to give pure product (6.75 g, 50%).

Androsta-4, 16-dien-3-one (25) A solution of 10 g of Androsta-5,16-dien-3/3-ol (24) in 475 cc of toluene and 75 cc of cyclohexanone was distilled (ca. 50 cc of distillate was collected) to eliminate moisture, 5 g of Al(OPr')3 10 m 50 cc of toluene was added and the solution was refluxed for 1 hour. Water then was added, volatile components were removed by steam distillation and the residue was extracted with chloroform. Evaporation of the dried extract, followed by crystallization of 15 the residue from chloroform-hexane, yielded 7.53 g of Androsta-4,l6-dien-3-one (25). Another 0.97 g (total, 8.5 g, 8 6%) was obtained by chromatography of the mother liquor on neutral alumina.

Example 6 - Synthesis of Androsta-3,5.16-trien-3-vl 2 0 methyl ether C12).

To a partial solution of androsta-4,16-dien-3-one (1.00 g, 3.70 mmol) in 2.2-dimethoxypropane (5.0 mL, 41 mmol) and 5 mL DMF were added methanol (0.2 mL) and p-toluenesulfonic acid monohydrate (26.4 mg, 0.139 mmol) The mixture was re fluxed & h, after which it, war. cooled and sodium bicarbonate (152.5 mg) was added. The suspension was partitioned between 50 raL of ice water and 50 mL of ethyl acetate. The organic layer was washed with two 50 mL portions of water + 5 0 mL of brine, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residual oil was taken up in 50 mL of hot hexane and filtered through a 12 mm x 30 mm column of silica gel 60 using 150 mL of hot hexane. The combined filtrates were concentrated under reduced pressure and recrystallized from acetone/methanol to give white crystals (468.0 mg, 1.645 mmol, 44%), m.p. 83-92°C.

Example 7 - Synthesis of 17-methvlene-Androst-4-en-ols.

To 20-homoandrosta-4,17-dien-3-one (119.0 mg, 0.4184 mmol) in 5 mL of methanol were added sodium borohydride (6.0 mg, 0.16 mmol) and 77 /j.L of water. After stirring 2 h further sodium borohydride (32.0 mg, 0.846 mmol) was added and the mixture was stirred overnight. After concentrating under reduced pressure the residue was purified by preparative TLC (5% ethyl acetate/hexane on silica gel) to give a more polar (59 8 mg) and a less polar (1.7 mg) product.

Example 8 - Synthesis of 17-methvlene-6-oxo-Androsta-4-en-3-one.

To a cooled solution of 2 0-homoandrosta-5,17-dien-3-ol (399.4 mg, 1.394 mmol) m 50 mL of acetone was added 2.67M Jones reagent (2.0 mL, 5.3 mmol). After stirring 1 h tne reaction was quenched with isopropanol (1.0 mL, 13 mmol) and poured into 100 mL of water The mixture was extracted three times with 50 mL portions of ethyl acetate and the combined organic extracts were washed with 50 mL of saturated sodium bicarbonate +■ 50 mL of brine. The organic phase was then dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was recrystallized from 95% ethanol to give an almost white powder (177.8 mg, 0.5958 mmol, 43%), m.p. 113-115°C.

Example 9 - Synthesis of 6B-OH-Androsta-4.16-dien-3-one.

To a solution of androsta-3,5,16-trien-3-yl methyl ether, (12) (200.5 mg, 0.7049 mmol), in 5 mL of 1,2-dimethoxyethane (DME) and 1 mL of water was added m-chloroperbenzoic acid (MCPBA, 77.4%, 173.2 mg, 0 776 mmol) suspended in 5 mL of DME + l mL of water + 0.40 g of 5% (w/w) NaOH dropwise, with stirring, over a period of 90 mm After stirring 18 h further MCPBA (247.0 mg, 1.11 mmol) suspended m 10 5 mL of DME + 2 mL of water + 0.8 g of 5% (w/w) NaOH was added dropwise, with stirring, over 1 1/2 h. The reaction mixture was stirred 1/2 h and then poured into 25 mL of saturated sodium bicarbonate. The aqueous mixture was extracted three times with 25 mL 10 of ether and the combined organic extracts were washed with 50 g of 5% (w/w) sodium thiosulfate + three 50 mL portions of brine, dried over magnesium sulfate, filtered through Celite, and concentrated under reduced pressure. The resulting crystalline 15 residue was purified by preparative TLC (35% ethyl acetate/hexane on silica gel) followed by two-fold recrystallization from aqueous ethanol to give lustrous white platelets (102.3 mg, 0 3571 mmol, 51%) , m.p 165-166 °C 2 0 Example 10 - Electrophvsioloqy of Androstane Stimulation of the Human VNO and Olfactory Epithelium.

A non-invasive method has been employed to record local electrical potentials from the human 25 vomeronasal organ (VNO) and from the olfactory epithelium (OE). Localized gaseous stimulation was applied to both nasal structures at different instances using specially designed catheter/electrodes connected to a multichannel drug 5 delivery system. This electrode and delivery system has been described by Monti and Grosser (J. Steroid Biochem. and Molec. Biol. (1991) .39:573 ) and m commonly owned, copending USSN 07/771,414, incorporated herein by reference. The local response 10 of the VNO and the OE showed a correlation with the concentration of the ligand stimulus The study was performed on ten clinically normal (screened) volunteers - 2 males and 8 females, ranging m age from 18 to 8 5 years. The studies were 15 conducted without general or local anesthetics.

The catheter/electrodes were designed to deliver a localized stimulus and simultaneously record the response. In the case of VNO recording, the right nasal fosa of the subject was explored 2 0 using a nasoscope (nasal specule) and the vomeronasal opening was localized close to the intersection of the anterior edge of the vomer and the nasal floor. The catheter/electrode was gently driven through the VNO-opening and the electrode tip placed in the 25 organ's lumen at 1 to 3 mm from the opening The nasoscope was then removed In the case of the OE, recordmg the procedure was similar except the positioning of the catheter/electrode was gently placed deep in the lateral part of the medial nasal duct, reaching the olfactory mucosa Localized gaseous stimulation was done through the catheter/electrode. A constant stream of clean, nonodorous, humidified air at room temperature was continuously passed through a channel of the stimulating system The stimulating ligand substances were diluted in propylene glycol, mixed with the humidified air, and puffed for from 1 to 2 seconds through the catheter/electrode. It is estimated that this administration provides about 25 pg of the steroid-ligand to the nasal cavity.

The results of this study are presented in Figures 4A, 4B, and 4C. The response is measured in millivolt-seconds (mV x s). Androsta-4,16-dien-3-one elicits a significantly stronger VNO response in females than do the other compounds tested (Fig. 4A). Furthermore, the VNO response to Androsta-4,16-dien-3-one is sexually dimorphic - twice as strong m females as it is in males (Fig. 4B). In contrast, the OE response in both males and females is low compared to a strong odorant such as clove (Fig. 4C).

Example 11 - Measurement of the Change m Receptor Potential of the Neuroepitihelium of the VNO m Response to Various Steroids.

The change m receptor potential m response to five different ligands was measured m 40 female (Fig. 5A) and 40 male (Fig. 5B) subjects.

Each subject was administered 60 pg of each of seven substances as indicated m the figure. The substances were administered separately for 1 second, using the procedure described in Example 10. The change m potential of the neuroepithelium of the VNO was recorded over time and the integral of the change in potential for each of the forty subjects was averaged. The results are shown m the figure. Comparison of Figures 5A and 5B show that each steroid is sexually dimorphic in its activity, and that some ligand substances are stronger in males while others are stronger in females.

Example 12 - Measurement of Autonomic Responses to 16-Androstene Stimulation of the VNO.

Various autonomic parameters were monitored as Androsta-4,16-dien-3-one was administered to 40 female subjects using the procedure described m Example 10 Propylene glycol was also administered as a control The ligand was administered as a 1 second pulse. The change in autonomic function was first noted within 2 seconds and lasted for up to 45 seconds. As shown in Figure 6, when compared to a propylene glycol control, the Androstane induced a 5 significant change in the integrated receptor potential in the VNO (6A), galvanic skin response (6B), skm temperature (6C), the percentage of cortical alpha wave activity as measured by electroencephalogram (6D) , peripheral arterial pulse 10 (6E), and respiratory frequency (6F).

Example 13 - Comparison of the Change in Receptor Potential Induced by Two Androstane Steroids. 60 picograms of each ligand steroid and of a propylene glycol control were administered to 5 15 female subjects as described in Example 10. As shown m Figure 7, Androsta-4,16-dien-3$-ol induced a greater change m receptor potential than did Androsta-4,16-dien-3-one.

Example 14 - Psychophysiological Effect of Androstane 2 0 Stimulation of the VNO.

The psychophysiological effect of Androstane stimulation of the VNO was measured by the coordinate administration of pheromone and questionnaire evaluation of the subject before and after administration The questionnaire included a panel of adjectives used as part of the standard Derogatis Sexual Inventory evaluation.

The subjects were 4 0 women between the ages 5 of 20 and 45, all in good health. The women were randomly assigned - 20 exposed to placebo and 20 exposed to about 20 picograms of Androsta-4,16-dien-3-one, administered as described m Example 10, supra. Subjects were given a 70 item questionnaire 10 evaluating feeling states immediately before and 30 minutes after administration of either placebo or experimental substance. The 70 adjectives of the questionnaire were randomly administered and subsequently clustered for evaluation based on their 15 relevance to each mood, feeling, or character trait. The results were as follows: Changes in feelings of social warmth, personal well-being, arousal/excitement, and aggression, from before administration to 3 0 minutes after administration, 20 were not significant in those exposed to the 16-Androstene compared to the changes resulting from administration of the control However, the decrease in negative affect (nervous, tense, ashamed, anxious, irritable, angry, enraged - T-test: p<0.0001, Anova: 25 p<0.04) , negative mood and character (sensitive, regretful, blameworthy, guilty, remorseful, sad, hopelQGs, resentful, worthless, miserable, unhappy, bitter, timid - T-test* p<0.0004, Anova p<0.06), and overall negativity (the combination of affect and character - T-test: p<0.0003, Anova: p<0.05) were 5 highly significant after 16-Androstene administration as compared to administration of the control.

Overall, these results suggest a sedative and/or anti-anxiety, and/or anti-depressant effect of Androsta-4 , 16-dien-3-one when administered 10 intranasally.

Example 15 - Treatment of Women for Premenstrual Stress.

Women experiencing the symptoms of premenstrual stress (PMS) are provided with a 15 pharmaceutical preparation of an Androstane steroid (preferably Androsta-4,16-dien-3-one, or Androsta-4, 16-dien-3a (/3) -ol) suitable for nasal administration. The steroid is provided as an ointment at a concentration of about 1 microgram/ml and about 0.1 20 ml is applied The ointment is applied just inside the nare of each nostril, three times daily. A similar method of treating PMS uses an aerosol preparation of the same steroid. The aerosol is sprayed into each nostril threes times daily.

Exarap]e 16 - Electrophysiological Studies The following electrophysiological studies were performed in 60 clinically normal human volunteers of both sexes (30 male and 30 female) 5 whose ages ranged from 20 to 45 years. No anesthetics were used, and female subjects were excluded if pregnant.

The stimulation and recording system consists of a "multifunctional miniprobe" described 10 elsewhere (Monti-Bloch, L. and Grosser, B.l. (1991) "Effect of putative pheromones on the electrical activity of the human vomeronasal organ and olfactory epithelium," J Steroid Biochem. Molec. Biol 39:573-582.). The recording electrode is a 0.3 mm silver 15 ball attached to a small (0 1 mm) silver wire insulated with Teflon© the surface of the electrode is first treated to produce a silver chloride interface, and is then covered with gelatin It is positioned within a small caliber Teflon® catheter 2 0 (dia = 5 mm) such that the tip of the electrode protrudes approximately 2 mm. The Teflon® catheter is 10 cm in length and constitutes the terminal extension for a multichannel delivery system which delivers a continuous air stream carrying discreet 25 pulses of chemosensory stimuli. The air stream first passes into a small chamber and is bubbled through a solution containing either a vomerophenn or an olfactant m a diluent or the diluent alone. A solenoid is used to rapidly redirect the air stream from the chamber to a route which bypasses the 5 chamber. This creates a discreet pulse of stimulant in the air stream. A second, outer Teflon® tube with a diameter of 2 mm surrounds the catheter-electrode assemblage, and its central end is connected to an aspirator that provides continuous suction of 3ral/s. 10 This concentric arrangement of the outer suction tube allows the emitted chemosensory stimuli to be localized to an area we call a "minifield" (approx. dia = 1 mm), and it avoids diffusion of substances either to the area outside the intended stimulation 15 site or into the respiratory system. The entire stimulating and recording assemblage may be positioned either on the neurosensory epithelium within the VNO, or on the surface of the olfactory or respiratory epithelium 20 Electro-voroeronasogram (EVG): Recordings are carried out m a quiet room with the subject supine; the multi-functional miniprobe is initially stabilized within the nasal cavity using a nasal retractor placed in the vestibule Reference and ground 25 electrodes consist of silver discs (8 mm), both of which are positioned on the glabella.

The entrance to the VNO, or vomeronasal pit, is identified by first dilating the nasal aperture and vestibule. A 6x magnifying binocular loupe with halogen illumination is then used to 5 introduce the tip of the Teflon® catheter and recording electrode assemblage into the VNO opening where it is stabilized at an approximate depth of 1 mm within the vomeronasal passage. Optimal placement of the recording electrode is signaled after testing 10 for an adequate depolarization in response to a test substance.

Electrical signals from the recording electrode are fed to a DC amplifier after which they are digitized, computer monitored, and stored. The 15 peak-to-peak amplitude of the signals is measured, and the area under the depolarization wave is integrated, while continuously monitoring the signal both on the computer screen and on a digital oscilloscope. Artifacts produced by respiratory 20 movements are deleted by training the subjects to practice mouth breathing with velopharyngeal closure. Chemosensory Stimulants Olfactory test substances are cineole, and 1-carvone; vomeropherins are A, B, C, D, E and F. Samples of vomeropherins in 25 concentration of 25-800 fmoles are delivered in the continuous air stream for durations from 3 00 milliseconds to 1 second. Usually, intervals of 3 to 5 minutes separated each series of short test pulses. All components of the lines carrying the test stimuli are made of Teflon®, glass or stainless steel and are 5 carefully cleaned and sterilized before each use. Electro-olfactcrram (EOG) : Olfactory recordings employed the same stimulating and recording multifunctional miniprobe as that used for the VNO. The tip was slowly introduced until the recording 10 electrode touched the olfactory mucosa. Adequate placement was signaled by a depolarization in response to a pulse of the odorant test substance.

Cortical evoked activity was induced by VNO stimulation with vomeropherins, and olfactory 15 stimulation with odorants delivered in 300 ms air pulses. It was recorded using standard electroencephalographic (EEG) electrodes placed at positions Cz-Al and Tz-Al of the international 10120 system; the ground electrode was placed on the 20 mastoid process. Electrodermal activity (EDA) was recorded using standard 8 mm silver electrodes in contact with palmar skin of the medial and ring fingers respectively, through a conductive gel interface. Skin temperature (ST) was recorded by a 25 small (1.0 nun) thermistor probe placed in the right ear lobe. Peripheral arterial pulse (PAP) was monitored with a plethysmograph attached to the tip of the index finger. Respiratory frequency (RF) was measured with an adjustable strain gauge placod around the lower thorax. All electrical signals were 5 DC amplified, digitized (MP-100, Biopac Systems) and continuously monitored utilizing a computer. Statistical Analysis: EVGs or EOGS, peak-to-peak changes and frequency changes of other parameters were measured and statistically analyzed. The 10 significance of the results was determined by either using paired t-tests or analysis of variance (ANOVA). Effect of Vomeropherins on the EVG: Each of the vomeropherins was found to produce a sexually dimorphic receptor potential (Fig. 8A-B). Recordings 15 of the EVG were performed on 30 men and 30 women (ages 2 0 to 45). Vomeropherins were diluted and applied as 1 second pulses to the VNO with b minute intervals between pulses when questioned, the subjects were not able to "smell" or otherwise 20 consciously detect any of the vomeropherins. This finding is in agreement with results previously reported (Monti-Bloch, L. and Grosser, B.l. (1991) "Effect of putative pheromones on the electrical activity of the human vomeronasal organ and olfactory 25 epithelium," J Steroid Biochem Molec Biol. 39:57 3-582.) which indicated that neither olfactory nor vomoropherin test stimuli delivered to the VNO elicit a perceptible sensation at the delivered concentration.

Fig. 8A shows the average response of male 5 subjects (ages 20 to 38) to the diluent, and to equimolar quantities (100 fmoles) of five vomeropherins (A, B, C, D and F), and to E, a stereoisomer of F. The profile of the response to each of the substances was similar in all subjects 10 regardless of age, and ro significant differences were revealed either by t-tests or by analysis of variance. For example, A, C and D produced significant effects (M,s = 11.4 mV, SD = 3.6 mV; M76 = 6.4 mV, SD 2.5 mV, and = 15.1 mV, SD = 4.9mV; 15 p<0.01), that were consistent in all individual cases. Other vomeropherins depolarized the VNO-receptors to a much lesser extent, but with consistent mean response amplitudes from individual to individual. Vomeropherins active m male subjects 20 produced larger responses than the diluent (p<0.00l). B, F and similar concentrations of olfactants induced significantly reduced responses in the male VNO (Fig. 8A and Fig. 9).

A similar experimental protocol was 25 followed with the 30 female subjects (ages 20 - 45). Among the vomeropherins, F (100 fmoles) produced the most significant differences within the group (Fig. 8B). Here, A induced a small effect that was significantly different from F (p<0.01). In both populations of subjects, active vomeropherins induced 5 receptor responses having large standard deviations (Fig. 8) . When the frequency distribution of the effects of A and F was studied in males and females respectively, we found a bimodal distribution. The significance of this observation is being studied at 10 this point.

E, a stereoisomer of F, does not stimulate the VNO in female subjects while F does (Fig. 8B). This is a demonstration of the specificity of VNO recognition of vomeropherins. In this regard it is 15 interesting to note that while F is a superior vomeropherm, E generates a stronger olfactory effect than does F (Fig. 8B and Fig. 9).

Effects of Vomeropherins on the EOG The summated receptor potential from the olfactory epithelium (OE) 20 was recorded in 20 subjects: 10 males and 10 females. In contrast to the sensitivity of the VNO to vomeropherins, the OE is less sensitive to these substances. This is true for both males and females (Fig. 9A). The mean receptor potential amplitude 25 ranged from 2.3 mV to 0.7 8 mV. In this study, B was the only vomeropherm having significant effect m the OE (p<0.02). Of the subjects questioned about odorant sensations following each stimulus presentation, 16 reported no olfactory sensation, while three males and one female described B as an unpleasant odor. This finding reveals that at the concentrations used in our study, most vomeropherins are not effective stimulants of the olfactory receptors, but do have a clear effect on vomeronasal receptors.

Effects of Olfactants on the EVG and EOG: In contrast to vomeropherins, the olfactants 1-carvone and cineole produce only a minor local response m the VNO (Fig. 9B). This was true for both men and women. As expected, these olfactants produced a strong response in both men and women (p<0.01) when locally applied to the OE (Fig. 9A). The diluent depolarized olfactory receptors to a lesser extent than cineole or 1-carvone (p<0.01), and it did not produce an olfactory sensation.

Reflex Effects of Vomeropherins: Studies were conducted to determine the central nervous system (CNS) reflex responses to vomeropherm stimulation of the VNO. The sexually dimorphic local responses induced by vomeropherins (Fig. 8A and B) were mirrored in the autonomic response of male & female subjects. In male subjects (Fig. 8C), A and C decreased skin resistance (electrodermal activity = EDA) (p<0.01, n = 30). In female subjects. (Fig. 8B), F and B produced greater decrease in EDA than A or C (p<0.01, n = 30).

Vomeropherins A and C induced a significant increase in skin temperature (ST) (Pig. 8G) in 30 male subjects (p<0.01); however D induced significant temperature decrease (p<0.01). In 30 female subjects (Fig. 8H) B and F evoked a significant increase in skin temperature (ST) (p<0.01) compared to A and C. In female subjects vomeropherins produced changes m EDA and ST with a greater standard deviation than in males.

Cortical activity was recorded from Cz and Tz in male and female subjects during application to the VNO of air pulses (300 ms to 1 sec) containing 200 fmoles of vomeropherm (Fig. 8G and H). In males (Fig 8E) A, C and D significantly increased alpha cortical activity with a latency of 270-380 ms. D and A evoked the strongest effect (p< 0.01) Synchronization of the EEG was sustained for 1 5 to 2.7 minutes after application of a single pulse of active substance. In females (Fig. 8F), a single pulse (200 fmoles) of B or F applied to the VNO increased alpha cortical independent of the response of olfactory receptors We found characteristic speciflcities m the response of the human VNO and the olfactory epithelium which suggests that they are independent functional systems with separate connections to the CNS (Brookover, C (1914) The 5 nervus termmalis in adult man. J. Comp. Neurol. 2^4:131—135.) There is also preliminary evidence that the EVG is not associated with trigeminal nociceptor endings since application of a local anesthetic (2% lidocaine) to the respiratory epithelium of the nasal 10 septum neither blocks nor diminishes the EVG (Monti-Bloch, L. and Grosser, B.l. (1991) "Effect of putative pheromones on the electrical activity of the human vomeronasal organ and olfactory epithelium," J. Steroid Biochem. Molec. Biol 39:573-582.). also, 15 subjects failed to report sensations of pain as a consequence of any of the stimulation procedures.

VNO receptors are clearly more sensitive to vomeropherins than to any of the olfactants tested; the opposite is true for olfactory receptors While 2 0 the OE may have receptor sites for some vomeropherins, the response specificity of the VNO is clearly different Sexual differences were noted in the specificities and effects of two groups of 25 vomeropherins, A, C and D; and B and F. This suggests a possible receptor-related sexual dimorphism. The findings suggest the activation of components of the autonomic nervous system in the adult human by vomeropherin stimulation of the VNO.

Furthermore, the results suggest that 5 stimulation of the VNO with vomeropherins produces synchronization of the EEG (Fig. 8G and H) Thus, the evidence herein indicates that the vomeronasal system responds to a variety of chemosensory stimuli, and that some are able to induce reflex autonomic 10 activity. 70

Claims (4)

WHAT WE CLAIM IS:

1. A use, in the preparation of a medicament for altering a hypothalamic function of an individual, of a ligand for a chemoreceptor displayed on the surface of nasal neuroepithelial cell of said individual wherein said cell is a part of tissue other than olfactory epithelia

2 A use, in the preparation of a medicament for altering an autonomic function of an individual, of a ligand for a chemoreceptor of a nasal neuroepithelial cell of said individual wherein said cell is a part of tissue other than olfactory epithelia

3 The use of Claim. 1 or Claim 2 wherein said neuroepithelial cell is located within a vomeronasal organ of said individual

4 The use of claim 3 wherein said ligand comprises an Androstane steroid

5. The use of claim 4 wherein said

Androstane steroid has the formula pNTELLECTUAi PROPERTY OFFIC OF N Z

3 0 JUL 1999

71

7

V

"7

■ u wherein P, is selected from the group consisting of oxo, a-(/?-) hydroxy, a-(/3-) acetoxy, a-(/?-) propionoxy, a-(j3-) methoxy, ar-(£-) lower acyloxy, a-(/3-) lower alkyloxy, and a-(/3-) benzoyloxy; P2 is selected from the group consisting of hydroxymethyl, acyloxymethyl,

alkoxymethyl, lower alkyl, hydroxyalkyl,

acyloxyalkyl, and alkoxylalky1 / P3 is absent or is selected from the group consisting of hydroxymethyl, acyloxymethyl, alkoxymethyl, lower alkyl, hydroxyalkyl, acyloxyalkyl, and alkoxylalkyl/ P4 is selected from the group consisting of hydrogen, oxo, halo, hydroxy, alkoxy, and acyloxy; P3 represents one or 2 substituents, wherein P5 comprises one or two hydrogen atoms, methyl, methylene, or one or two halo atoms; P4 is hydrogen or halo; and "a", "b", "c", "d", "e", "f", and "h" are alternative sites for optional double bonds.

6. A use P3 is lower alkyl.

according to Claim 5 wherein

7. A use "b" is a double bond.

according to Claim 5 wherein

A use according to Claim 7 wherein

P( is oxo,

■J -LL(.rTtjAL IT-,: _aiv offiii if >>z

3 0 JUL 1999

hbCElVED

i

7 2 1 *

9. A use I according to Claim 8 wherein P4 is hydrogen.

10- A use according to Claim 9 wherein said steroid is selected from the group consisting of androsta-4,l6-dien-3-one, androst-4-en-3-one, 17-methylene-androst-4-en-3-one, 18-nor-17-methyl-androsta-4,17(13)-dien-3-one, 19-hydroxy-androsta-4,16-dien-3-one, 19-hydroxy-androst-4-en-3-one, 19-hydroxy-17-methylene-androst-4-en-3-one and 18-nor-17-methyl-19-hydroxy-androsta-4,17(13) -dien-3-one.

11* A use according to Claim 8 wherein

P4 is oxo.

12. A use according to Claim 11 wherein said steroid is selected from the group consisting of androsta-4,16-dien-3,6-dione, androst-4-en-3,6-dione, 17-methylene-androst-4-en-3,6-dione and 18-nor-17-methy1-androsta-4,17(13)-dien-3,6-dione.

13. A use according to Claim 8 wherein P4 is hydroxy.

14 A use according to Claim 13 wherein said steroid is selected from the group consisting of 6a-hydroxy-androsta-4,16-dien-3-one, 6a-hydroxy-androst-4-en-3-one, 17-methylene-6a-hydroxy-androst-4-en-3-one and 18-nor-17-methyl-6a-hydroxy-androsta-4,17(13)-dien-3-one.

15. A use according to Claim 7 wherein Vl is hydroxy.

16. A use according to Claim 15 wherein said steroid is selected from the group consisting of androsta-4,16-dien-3a-ol, androsta-4, 16-dien-3S-ol,

intellectual property office"i

OF ti Z

3 0 JUL 1999

T T f* '

7 3 ^ J t u „

androst-4-en-3a-ol, androst-4-en-3S-ol, 17-methylene-androst-4-en-3a-ol, 17~msthylene-androst-4-en-3fl-ol, 18-nor-l7-methyl-androsta-4, 17(13)-dien-3a-ol, 18-nor-17-methyl-androsta-4, 17(13)-dien-3 6-ol.

17. a use according to Claim 7 wherein "e" or "d" is a double bond.

18 A use according to Claim 1 or Claim 2 wherein said steroid is selected from the group consisting of androsta-4, 6,lS-trien-3-one, androsta-4,6-dien-3-one, l7-raethylene-androsta-4, 16-dien-3-one, 18-nor-l7-methyl-androsta-4, 6,17(13) -tnen-3-one, androsta-1,4,16-trien-3-one, androsta-1, 4-dien-3-one, 17-methylene-1,4-dien-3-one, 18-nor-17-methyl-androsta-1, 4 ,17(13) -tnen-3-one, androsta-1, 4 ,16-trien-3a-ol, androsta-1,4-dien-3a-ol, 17-methylene-androsta-l, 4-dien-3a-ol, 18-nor-17-methyl-androsta-l, 4,17(13)-trien-3a-ol.

19 a use according to Claim 5 wherein "c" is a double bond.

20 A use according to Claim 19 therein said steroid is selected from the group consisting of androsta-5(6),16-dien-3a-ol, androst-5(6)-en-3a-ol, 17-methylene-androst-5(6)-en-3a-ol, 18-nor-17-methyl-androsta-5(6),17(13)-dien-3a-ol.

21. A use according to Claim 5 wherein "a" is a double bond.

22. a use according to Claim 21 wherein said steroid is selected from the group consisting of

3-methoxy-androsta-3 , 5 (6) ,16-triene, 3-xnethoxy-

androsta-3,5(6)-diene, 17-methylene-androsta-3,5(6)-

INTELLECTIJAL prl'perlt ofhce f of n 2 |

0 0 JUL 1999 j

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diene, 18-nor-17-methyl-3~methoxy-androsta-3,5(6),17(13)-tnene.

23. A use according to Claim 5 wherein P3 is methyl.

24. A use according to Claim 23 wherein Pj is methylene.

25. A use according to Claim 24 wherein said steroid is selected from the group consiting of 17-methylene-androst-4-en-3-one, 17-methylene-androst-5 (6) -en-3a-ol, 17-methylene-androst-4-en-3a-ol, l7-methylene-androst-4-en-3B-ol, 17-methylene-androsta-4,6-dien-3-one, 17-methylene-androst-4-en-

3,6-dione, 17-methylene-19-hydroxy-androst-4-en-3-one, l7-methylene-3-inethoxy-androsta-3,5(6)-diene, 17-methylene-androsta-l;4-dien-3-one, 17-methylene-androsta-l , 4-dien-3a-ol, 17-raethylene-6a-hydroxy-androst-4-en-3-one.

26. a use according to Claim 23 wherein Pj is two hydrogen atoms.

27. A use according to Claim 26 wherein said steroid is selected from the group consisting of androst-4-en-3-one, androst-5(6)-en-3a-ol, androst-4-en-3a-ol, androst-4-en-3B-ol, androsta-4, 6-dien-3-one, androst-4-en-3,6-dione, 19-hydroxy-androst-4-en-3-one, 3-methoxy-androsta-3,5(6)-diene, androsta-1,4-dien-3-one, androsta-1,4-dien-3a-ol, androst-4-en-6a-ol-3-one.

28- A use according to Claim 5 wherein P3 is methyl and "h" is a double bond.

LLICTIV r ,

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3 0 JUL 1933

RPPCn/cn

29. A use according to Claim 28 wherein said steroid is selected from the group consisting of androsta-4,l6-dien-3-one, 3a-hydroxy-androsta-5(6),16-diene, 3a-hydroxy-androsta-4,16-diene, 30-hydroxy-androsta-4,16-diene, androsta-4,6,16-trien-3-one, androsta-4,16-dien-3,6-dione, 19-hydroxy-androsta-4,16-dien-3-one, 3-methoxy-androsta-3,5(6),16-triene, androsta-1,4,16-trien-3-one, 3a-hydroxy-androsta-1,4,16-triene, androsta-4,16-dien-6a-ol-3-one.

30 A use according to Claim 5 wherein Pj is methyl and "f" is a double bond.

31 A use according to Claim 4 wherein said steroid is selected from the group consisteing of l8-nor-17-methyl-androsta-4,17(13)-dien-3-one, 18-nor-17-methyl-androsta-5(6),17(13)-dien-3a-ol, l8-nor-17-methyl-androsta-4,17(13)-dien-3 Q-ol, 18-nor-17-methyl-androsta-4,17(13)-dien-36-ol, 18-nor-17-methyl-androsta-4,6,17(13)-trien-3-one, 18-nor-17-methyl-androsta-4,17(l3)-dien-3,6-dione, 18-nor-17-methyl-19-hydroxy-androsta-4,17(13)-dien-3-one, 18-nor-17-methyl-3-raethoxy-androsta-

3,5(6),17(13)-triene, l8-nor-17-methyl-androsta-1,4,17(13)-trien-3-one, 18-nor-17-methyl-androsta-1,4-dien-3a-ol and 18-nor-17-methyl-androsta-4,17(13)-dien-6a-ol-3-one.

32 A use according to Claim 16 wherein said steroid is androsta-4,16-dien-3B-ol

33 The use of any one of Claims 1 through 32 wherein the medicament is formulated for administration of least substantially 100 picograms, but no more than substantially 100 micrograms of said ligand

34

The use of Claim 33 wherein the medicament l formulated for administration of at least l^fc^^K^txiJally< 1

nanograms, but no more than substantially said ligand.

(ip M7

0 micrograms of

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J 0 JUL

1939

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35, The use of Claim 33 wherein the medicament is formulated for administration of at least substantially 10 nanograms, but no more than substantially 1 microgram of said ligand.

36. The use of any one of Claims 1 through 35 wherein the medicament further comprises said ligand dissolved m a pharmaceutically acceptable carrier

37 The use of Claim 36 wherein said medicament is an ointment.

38 The use of Claim 36 wherein said medicament is liquid

39 The use of Claim 36 wherein the medicament is formulated for administration by aerosol

40 The use of any one of Claims 1 through 39 wherein the medicament is formulated for administration of more than one Androstane steroia

41 The use of any one of Claims 1 through 40 wherein said medicament is formulated for co-administration nasally to said individual of an Estrene steroid.

42 The use of any one of Claims 1 through 41 wherein said function is the diminution of negative affect.

43 The use of any one of Claims 1 through 42 wherein said individual is a woman u'J ill,1 T I ,1Y iji n- , 12

3 0 1339

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44 The use ot Claim 43 wherein said alteration ot function results in a reduction of premenstrual stress in women.

45, The use of any one of Claims 1 through 43 wherein said function is the alleviation of the symptoms of psychoses, depression or anxiety

4 6 A use of 17-methyleneandrost-4-en-3a-ol in the preparation of a medicament for use m treating an.\iety.

47. A use of 17-methyleneandrost-4-en-33-ol in the preparation of a medicament for use in treating anxiety.

48 A use of androsta-4, 6, 16-tnen-3-one in the preparation of a medicament for use in treating anxiety.

49 A use of androsta-4, 16-dien-3(3-ol in the preparation of a medicament for use in treating anxiety

50 A use of androsta-5, 16-dien-3(3, 19-diol in the preparation of a medicament for use m treating anxiety

51 A use as defined in Claim 1 or Claim 2 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings

52 A use as claimed in any one of Claims 46 to 50 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings

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