WO2001039766A2

WO2001039766A2 - Screening invertebrate pheromones for therapeutic activity

Info

Publication number: WO2001039766A2
Application number: PCT/US2000/032363
Authority: WO
Inventors: Nadar Zamir; Yaakov Lavie
Original assignee: ALYDAR PHARMACEUTICALS Ltd; ALYDAR PHARMACEUTICALS Inc
Current assignee: ALYDAR PHARMACEUTICALS Ltd; ALYDAR PHARMACEUTICALS Inc
Priority date: 1999-12-01
Filing date: 2000-11-28
Publication date: 2001-06-07
Anticipated expiration: 2002-06-01
Also published as: AU1929401A; WO2001039766A3

Abstract

A method of screening pheromone compounds from a library of the same for therapeutic activity and use of such compounds in pharmaceutical compositions for the prevention and/or treatment of a variety of diseases, conditions and symptoms thereof including cardiac arrhythmia, multi-drug resistance (MDR) in cancer cells and tumors and microorganisms and diseases, conditions and symptoms associated with modulation of cGMP levels.

Description

METHOD FOR SCREENING NON-PEPTIDE, NON-STEROID INVERTEBRATE

PHEROMONES, COMPOSITIONS CONTAINING THE SAME AND USE OF THE

COMPOSITIONS TO TREAT DISEASES, CONDITIONS, AND SYMPTOMS

THEREOF

Field of the Invention

The present invention relates to pheromones and derivatives and analogs

thereof, more particularly to pheromones, derivatives and analogs thereof possessing

therapeutic activity in warm-blooded animals.

List of References

The following is a list of documents which are intended for a better

understanding of the present invention:

Bosch, I. and Croop, J., Biochimica et Biophysica Acta, 1288: F37-F54, 1996;

Burlakov, S.D. et al., International Application Publication No. 9727845, 1997;

Cam , A.J. and Yap, J.G., Cardiovasc. Electrophysiol., 10: 307-317, 1999;

Chinkers, M. et al., Nature, 338: 78, 1989;

Cohen-Tannoudji, J. et al., Physiology and Behavior, 56: 955-961 , 1994;

Danty, E. et al., C.R, Acad. Sci. Paris, 317: 1073, 1994;

Ding, K.H. et al, Cell Signal, __V. 87-94, 1999; Ding, K.H. et al., Life Sciences, 64: 161-174, 1999;

Fulle, H.J. et al., PNAS, 92: 3571 , 1995;

Hardemann, M.R. et al., Clinical Hemorheology, 14: 605-619, 1994;

Hobbs, A.J. et al., Annu. Rev. Pharmacol. Toxicol., 39: 191 , 1999;

Horikawa, K. et al., J.Exp. Med., 174: 1385-1391 , 1997;

Ishii et al., Am. J. Physiol. 256: C495, 1989;

Jacobson, M., U.S. Patent 2,900,756;

Juilfs, D.M. et al., PNAS, 94: 3388, 1997;

Kelly, D.R., Chemistry and Biology, 3: 595, 1996;

Kurokawa. ef a/., JP 01153610;

Leal, W.S. et al., Nature, 385: 213, 1997;

Nachshon, S. et al., Am. J. Physiol., 268: E.428, 1995;

Nakamura et al., U.S. Patent No. 5,190,978, 1993;

Namita et al., JP No. 01216921 , 1989;

Nikaido, H., Current Opinion in Microbiology, 1: 516, 1998;

Noula, C. et al., Biochem. Soc. Trans., 24: 303, 1996;

Rasmussen, L.E.L. et al., Nature, 379: 684, 1996;

Regnier, F.E. and Law, E.H., Journal of Lipid Research, 9: 541 , 1968;

Roberts, G.D., Goodman, N.L. et al., J. Clin. Microbiol. 18: 689, 1983;

Roden, D.M., In: Goodman and Gilman's The pharmacological basis of

therapeutics, 9^th ed., Hardin, J.G. and Limbird, L.E. (eds), 839-874, 1996;

Roelofs, W. L, PNAS, 92: 44-49, 1995; Sonneveld, P. et al., Current Opinion in Biotechnology, 9: 543-548, 1997;

Szekeres, L., and Papp J.G., Handbook of Experimental Pharmacology, Vol.

XVI/4, pp. 131-182, 1975; and

Walker, M.J.A., et al., The Lambeth Conventions: guidelines for the study of

arrhythmia's in ischemia, infarction and reperfusion. Cardiovascular Research, 22: 447-

455, 1988.

Wilson, E., Pharmacol. Ther., 24: 401-433, 1984;

Van Veen, H.W. et al., Nature, 15: 291-297, 1988;

Zamir, N. et al. Biochem. Biophys. Res. Comm.. 188: 1003, 1992;

Zamir, N. et al. Biochem. Biophys. Res. Comm., 197: 116, 1993; and

Ziegelsberger, G. et al., The Journal of Neuroscience, 10: 1217, 1990.

The entire content of each document cited above is incorporated herein by

reference in its entirety to the extent that no portion of the content incorporated is in

conflict with this specification. Subsequent reference to the above-mentioned

documents will be made by first author's name and in some cases year of publication.

Background of the Invention

Pheromones are a class of chemicals that are communicative between animals

of the same species and elicit stereotypical behavioral and endocrine responses

(Regnier, 1968). Although there is a huge variety of pheromone chemical structures, a single pheromone molecule has been shown to have biological effects in non-

associated species such as a species of insects and elephants (Rasmussen, L.E.L. et

al, 1996). Some chemical structure similarities exist between pheromones of different

species.

The pheromone communication system basically involves the release of specific

chemicals from a pheromone producer (emitter), transmission of these chemicals in the

environment to a receiver and the processing of the signals to mediate the appropriate

behavioral responses in the receiver (Roelofs, 1995). Most vertebrate pheromones are

believed to activate the vomeronasal organ (VNO) which resides anterior to the main

olfactory epithelium (MOE) in a blind-ended pouch within the septum of the nose. In

both the VNO and the MOE, neuroepithelial dendrites terminate in specialized cilia

containing specific receptors that bind odorants. This binding initiates a cascade of

enzymatic reactions that results in the production of second messengers and the

eventual depolarization of the cell membrane. Functional and chemical analysis has

shown that many pheromones are volatile.

Several of the pheromone receptors have been shown to be conserved across

species (Chinkers et al., 1989). Although the ligands that activate the pheromone

receptors in different species are different, similar chemical structures have been found

in such ligands originating from diverse species (Kelly, 1996) indicating a high degree

of conservation across species of the pheromones themselves. The signal transduction mechanisms through which pheromones exert their

effect involve various pathways which are commonly found in various other biological

processes. The pheromone signal transduction mechanisms have also been shown

to be conserved across species (Danty et al., 1994).

Recently, a medicinal alkaloid compound which was synthesized overforty years

ago, was isolated from a natural source and then identified as being an insect

pheromone (Leal W.S. et al., 1997).

In addition, an insect pheromone was described as useful for controlling

sensitivity of somatic cells of mammals to glucocortoid hormones (Burlakov et al.,

1997). The diol 1 ,2-hexadecanediol was described as an insect pheromone which is

extracted from virgin female moths and is an attractant for the male gypsy moth

(Jacobson, M., 1959). 1 ,2-hexadecanediol was shown to have anti-cancer and anti-

inflammatory activities (Nakamura et al., 1993 and Namita et al., 1989, respectively).

U.S. Patent No. 2,900,756 (Jacobson Martin, 1959) describes the diol 1 ,2-

hexadecanediol as an insect pheromone which is extracted from virgin female moths

and is an attractant for the male gypsy moth. 1 ,2-hexadecanediol was also shown to

be a component of a ram pheromone (Cohen-Tannoudji, J., 1994). 1 ,2-hexadecanediol was described as being useful in the prevention of cancer

in combination with cancer preventing agents (Nakamura et al., 1993). 1 ,2-

hexadecanediol purified from esterolysates of sheep wool wax showed some

cytotoxicity against Ehrilich Ascites Carcinoma (EAC) cells (Miwa, N., etal., 1997). 1 ,2-

hexadecanediol was also shown to have anti-inflammatory activity (Namita etal. , 1989),

as an inhibitor of phospholipase A2 (Noula, C, et al., 1996), as a regulator of iodine

metabolism in the thyroid gland (Van den Bergen et al., 1999) and used in the field of

cosmetics (Kurokawa, et al.).

Other hexadecanediols and some related compounds were also found to be

pheromones or components of pheromones such as, for example, 1 ,2-octadecanediol

involved in pheromone activity of rams together with 1 ,2-hexadecanediol and other

components, (Cohen-Tannoudji, J., 1994).

Arrhythmias are abnormal heart rhythms. Normally, the heartbeat originates in

the right atrium from an electric signal propagated by a group of specialized cells called

the sinoatrial (SA) node. The signal spreads throughout the atria and to the

atrioventricular (A-V) node. The A-V node connects to a group of specialized fibers in

the ventricles which conduct the electric signal to all parts of the ventricles. This route

of the electrical impulse through the heart enables it to pump properly (Roden, D.M.,

1996). An arrhythmia may occur as a result of disturbance in the normal sequence of

impulse initiation and propagation. Failure of impulse initiation may result in slow heart

rates (termed "bradycardia") and failure of impulses to propagate normally from the

atrium to the ventricle results in dropped beats or "heart block". These abnormalities

result mainly from use of various drugs or by structural heart disease. Bradycardia is

commonly treated by implanting an electronic pacemaker under the skin for elevating

the heart rate.

Rapid heart rate (termed "tachycardia") results in ineffective blood circulation.

Rapid heart rate arising from the ventricles (termed "ventricle tachycardia" VT)) may

be a life threatening situation.

The most serious cardiac rhythm disturbance is ventricular fibrillation (VF), a

condition in which the quivering of the lower ventricles of the heart adversely affect the

pumping of blood which may result in collapse and sudden death. Atrial fibrillation (AF)

is a condition in which the two small upper atria of the heart quiver instead of engage

in normal beating. As a result the blood is not completely pumped out of the atria

resulting in pooling and clotting of the blood. When a blood clot occurs, it may become

lodged in an artery in the brain resulting in a stroke.

The main causes of arrhythmia include myocardial infarction (Ml), myocardial

ischemia, aberrant conduction pathways, thyroid disease, drugs, electrolyte disturbances, cardiomyopathy and myocarditis. Arrhythmias may also be induced

chemically, electrically and mechanically (See, Szekeres, et al., 1975; and Wilson,

1984).

Non-pharmacological therapy of cardiac arrhythmias (mainly bradycardia)

includes the use of permanent or temporary pacemakers which are inserted

subcutaneously and supply electric stimulation. Implantable electronic cardioverter-

defibrillators are today a common treatment for various kinds of tachycardia. Ablation

therapy is currently widely used for the treatment of some types of arrhythmias.

Many arrhythmias are treated with anti-arrhythmic drugs. These drugs have

been classified into various groups and sub-groups based on electrophysiological

effects (e.g. action potential) and on interactions with membrane receptors and ion

channels (See, Roden, D.M., 1996; and Camm, A.J., et al., 1999). Some examples of

commonly used anti-arrhythmic drugs are sodium channel blockers such as flecainide,

procainamide, quinidine, and propafenone, beta adrenoceptor antagonists (beta

blockers) such as propranolol and esmolol, drugs that prolong repolarization and the

cardiac refractory period such as d-sotalol, amiodarone, dofetilide, and ibutilide, and

calcium antagonists such as diltriazem and verapamil, adenosine, and digoxin.

Although arrhythmias (mainly super ventricular and ventricular arrhythmias) have

traditionally been treated with anti-arrhythmic drugs, it has recently been shown that many anti-arrhythmic drugs have multiple side effects including enhanced arrhythmia,

enhanced re-entry, negative hemodynamic effects and in extreme cases mortality.

While anti-arrhythmic drugs are still used as a part of a first line therapy for

treatment of certain arrhythmias, due to their side effects, their long-term use continues

to decline and there is continued search for new effective agents which can be used in

chronic treatment of arrhythmia.

One of the major treatment regimes in cancer involves administration of a range

of anti-cancer cytotoxic drugs to the patient. However, although chemotherapy

improves long term survival in cancer patients, this treatment fails in many cases due

to resistance of the cancer cells to a wide range of anti-cancer drugs, a phenomena

termed "multi-drug resistance (MDR)." (See, Sonneveld, etal., 1999, and Bosch, etal.,

1996).

MDR may be either intrinsic, causing a tumor to initially fail to respond to any of

a wide range of functionally and structurally unrelated anti-cancer drugs or may be

acquired.

There are various cellular mechanisms which have shown to be involved in MDR

of cancer cells. Among these cellular mechanisms is over-expression of P-glycoprotein

(P-gp) encoded by the MDR-1 gene. The activity of the P-gp protein causes major acceleration in the efflux of chemotherapeutic drugs outwardly from the inside of the

cells. Other cellular mechanisms include increased DNA repair via alterations in DNA

polymerase and DNA topoisomerase-ll enzyme activities; increased inactivation of

drugs due to over-expression of glutathione-S-transferase and elevated intracellular

glutathione concentrations; altered drug metabolism; altered cellular pharmaco-kinetics

of drug uptake; and decreased apoptotic response.

In addition, other MDR-like proteins have been isolated and shown to be over-

expressed in various tumor cell lines. To date, the MDR-protein family includes, for

example, P-gp, multi-drug resistance-associated protein (MRP), lung resistance-

associated protein (LRP), and breast-cancer resistance protein (BCRP). The MDR-

protein family belongs to the same ATP binding cassette (ABC) super-family of

transporters.

Intense efforts in basic and clinical research have attempted to identify strategies

leading to MDR inhibition in cancertherapy. Compounds which increase the sensitivity

of multi-drug resistance cells to cytotoxic agents are termed "MDR-chemosensitizers",

"MDR-reversal agents", "MDR-modulators" or "MDR-inhibitors". Such compounds

have been tested for their potency both in vitro (using MDR cell lines) as well as in vivo.

Indeed, various MDR- chemosensitizers which include compounds of diverse structure

and function have been characterized. However at the reported effective doses, the

drugs induced major side effects, (e.g. cardiac effects of verapamil and quinine derivatives, immunosuppressive action of cyclosporines, and neurotoxic effects of

calmodulin). Presently, there are no anti-MDR drugs which are effective at tolerable

doses.

Microorganisms have also developed resistance mechanisms to various

antibiotic and chemotherapeutic drugs (Nikaido, H., 1998). Thus, the efficacy of

commonly used antimicrobial agents has been greatly reduced. It has been shown that

many antibiotics induce transcription of genes that confer MDR to the treated

microorganisms. Recently, it has also been shown that various microorganisms have

efflux pump systems capable of excreting a wide spectrum of drugs outwardly from the

microorganisms and that these systems are very similar to the MDR pump of

mammalian cells found on cancer cells and human tumors. One such MDR protein

found in bacteria is the LmrA protein which mediates antibiotic resistance in bacteria

by extruding amphiphilic compounds from the inner leaflet of the cytoplasmic membrane

(Van Veen, H.W., 1998). The LmrA protein is an ATP-binding transporter which is

similar to the human MDR P-gp protein. This type of MDR resistance efflux pump is

known to be conserved from bacteria to man. Thus, drugs that are less efficiently

pumped out of microorganisms or capable of inhibiting the efflux pump or capable of

increasing outer membrane permeability of such microorganisms are highly desired.

Recently it has been shown that one of the pheromone receptors is guanylyl

cyclase D (GC-D) (Fulle H.J., 1995). GC receptors have been shown to be conserved across species (Chinkers M, 1989). Although the ligands that activate the pheromone

in such ligands originating from diverse species (Kelly D.R., 1996). Odorant receptors

are found mainly in olfactory tissue. However, it has been shown that GC linked

receptors are expressed also on a variety of other cell types such as cardiocytes

(Nachshon, S., 1995), erythrocytes (Zamir, N., 1992) and sperm cells (Zamir, N., 1993).

The signal transduction mechanisms through which pheromones exert their

processes. The pheromone signal transduction mechanisms have also been shown

to be conserved across species (Burlakov, S.D. et al., 1997).

One such pathway involves elevation of the level of intracellular cyclic GMP

(cGMP) in cells. The elevation of cGMP may generally be a result of one of the

following:

(i) Stimulation of particulate GC receptor which leads to the activation of its

catalytic domain and to the production of cGMP(Juilfs, 1997). There are

several endogenous ligands, being peptides or polypeptides, that exert

their biological effect via activation of specific membranal GC receptors.

Examples of such peptides are atrial natriuretic peptide (ANP), brain

natriuretic peptide (BNP), c-type natriuretic peptide (CNP), guanylin and

uroguanylin. Although such peptide ligands were discovered over a decade ago, as of today, there is only one GC peptide, a BNP ligand,

which is currently in clinical trials for acute congestive heart failure

(Natrecor™, Sciosnova, Mountain View, CA). The difficulty in using

peptide GC ligands in therapy is mainly due to the fact that they exhibit

a very short biological half-life in blood (only about 1-2 mins.), they must

be administered by continuous i.v. infusion which is inconvenient and

requires hospitalization of the treated individual and, in addition, the

peptide GC ligands are very costly,

(ii) Elevation of cGMP may also result by employing activators of soluble

guanylyl cyclase. Examples of such substances are nitric oxide (NO) and

carbon oxide (CO),

(iii) A further means of elevation of cGMP is by inhibition of

phosphodiesterase (PDE) (such as PDE2 or PDE5). The inhibition of

PDE results in the prevention of cGMP degradation. The inhibition of

PDE5 for example, is the basis of the action of the therapeutic agent

VIAGRA™ (Pfizer) which is administered orally,

(iv) Elevation of cGMP may also result by inhibition of the enzyme neutral

endopeptidase (NEP). NEP inactivates the natriuretic peptides capable

of activating particulate GC receptor and therefore its inhibition will

prolong the action of the activating peptides resulting in elevation of the

intracellular cGMP levels. In addition to the above, pheromones may also exert their effect through various

other pathways which do not necessarily involve the elevation of cGMP.

Recently, a medicinal alkaloid compound which was synthesized over forty years

ago, was isolated from a natural source and then identified as being an insect

pheromone (Leal W.S. et al., 1997). This compound, most probably, acts via a non-

cGMP mechanism.

In addition, an insect pheromone was described as useful for controlling

1997).

Summary of the Invention

In accordance with the present invention it has been realized that invertebrate-

derived, non-peptide and non-steroid pheromones present a source of compounds

which may have a wide variety of pharmaceutical utilities. In accordance with the

invention the compounds of potential pharmaceutical activity are selected from libraries

of such pheromones through a screening process and the selected compounds may

then be used for different desired pharmaceutical utilities. Furthermore, select

pheromones may have a high therapeutic index, and thus may be potent

pharmaceutical agents. In a first aspect of the present invention, there is provided a method of screening

compounds to obtain those having a desired therapeutic activity, comprising the steps of:

(a) providing a biological model for screening of compounds, said

model being predictive for the desired therapeutic activity;

(b) testing compounds from a library of compounds in said model, said

library comprising at least one invertebrate-derived, non- peptide and non-steroid

pheromone, or a derivative thereof; and

(c) selecting at least one of said compounds showing therapeutic

activity in said model.

The present invention also provides a method of selecting compounds having

a desired therapeutic activity, comprising the steps of:

(a) providing data relating to the three-dimensional (3D) structure of

at least a pharmacophore of a compound known to have the desired therapeutic

activity;

(b) providing a library of compounds, said library comprising at least

one invertebrate-derived, non-peptide and non-steroid pheromone, or a derivative

thereof; and

(c) analyzing the 3D structure of one or more compounds of said

library and selecting a compound having a domain with a 3D structure identical or

similar to the 3D structure of said pharmacophore. Optionally, the above method further comprises the step of testing a selected

compound in a relevant biological model which is predictive of the desired therapeutic

activity.

The term "therapeutic activity" as used herein means an activity of a compound

on specific target cells, target tissue or target organ, or an activity which achieves a

specific effect within a body, typically a therapeutic effect. The term "therapeutic

activity" further means an activity including a preventive effect which is manifested in

relatively low levels or concentration of the active compound, which is a concentration

which gives rise to a biological effect within the body not through a general systemic

effect but rather through a specific effect on specific targets within the body. Typically,

but not exclusively, the specific therapeutic activity is achieved through binding of said

compounds to specific receptors, which may be extracellular receptors, membranal

receptors or intracellular receptors. The term "therapeutic activity" shall include, but is

not limited to the inhibition of multiple drug resistance (MDR) in cancer cells and tumors

and pathogenic microorganisms, the elevation of ordecrease in cGMP levels, and anti-

arrhythmic activity.

A biological model suitable for screening compounds of interest herein may be

a model which is known and acceptable in the literature for screening compounds to

select those which have a desired effect. In some indications, the acceptable models

are in vitro models, e.g. models involving testing of the effect of the compound on a cell or tissue culture. In other cases, a relevant model having a predictive value is an in

vivo model involving laboratory animals, which may be rodents such as rats, mice or

rabbits; xenograft models, e.g. an immune-compromised mouse carrying human tissue;

higher order animals such as cats, dogs and even primates. One such typical animal

model is a model for determining anti-arrhythmic activity using rats in which arrhythmias

and myocardial infarctions are induced by coronary artery ligation. Furthermore, the

relevant model may at times be a combination of in vitro and in vivo models, or a

combination of several in vitro and/or several in vivo models. In addition, at times the

relevant model may also be a model of non-living material such as a model of isolated

membranes, a variety of biochemical assays, and the like.

Where the compound having the desired pharmaceutical activity is selected on

the basis of its 3D structure, the term "pharmacophore" should be understood as

meaning the structural domain on the compound which is associated with the desired

therapeutic activity. This domain may, for example, be the domain in the compound

which interacts with an enzyme involved in a biochemical pathway, a receptor to which

an active ligand binds, or any other cellular constituent.

The term "invertebrate pheromone" (or "pheromone") in the context of the

present invention should be understood as encompassing any chemical compound

isolated from any invertebrate species which is produced and discharged from glands

and external ducts and functions by influencing other members of the same species in one of the ways known in the art (see Regnier, et al., 1968). One example of an

invertebrate order are insects, one of the predominant species of insects being

Lepidoptera.

The term "pheromone" encompasses the natural pheromones as well as

synthetic compounds which display a similar activity. Such synthetic compounds

include various derivatives of the natural pheromones, as well as analogs which display

a desired activity.

The term "pheromone-containing composition" as used herein refers to

compositions suitable for administration to warm-blooded animals including humans to

prevent and/or treat a disease, condition or symptom thereof which contain at least one

pheromone.

The invention further provides a method of treating warm-blooded animals

including humans afflicted with a disease, a condition or symptoms associated

therewith, comprising administering to the individual an effective amount of an

invertebrate-derived, non-peptide, non-steroid pheromone as an active agent, the active

agent being selected by one or more of the screening methods of the invention for the

desired activity suitable for treating the disease, condition or symptoms thereof.

Methods of preventing disease, condition or symptoms thereof by the administration of

the active agent are also within the scope of the present invention. The present invention also provides an invertebrate-derived, non-peptide and

non-steroid pheromone having a desired therapeutic activity alone and in the form of

a pheromone-containing composition, said pheromone obtained by the above methods

of the invention. Such pheromones will be referred to herein at times as "pheromone

or pheromones of the present invention".

In accordance with the present invention it has been realized that since some of

the activities of invertebrate-derived, non-peptide and non-steroid pheromones possess

anti-arrhythmic activity, such pheromones are a source of compounds which may be

used for a variety of therapeutic uses in the treatment of diseases, conditions and

symptoms associated with cardiac arrhythmia. Compounds of the present invention

having potential therapeutic activity may be selected first on the basis of their ability to

reduce or treat cardiac arrhythmia. Such target compounds may be screened from a

library of pheromone compounds to obtain compounds having the desired therapeutic

uses.

the activities of invertebrate-derived, non-peptide and non-steroid pheromones are

manifested via cyclic guanosine monophosphate (cGMP) and since guanylyl cyclase

(GC) receptors have been shown to be relatively conserved between species, such

pheromones are a source of compounds which may be used for a variety of

pharmaceutical utilities in which there is an involvement of intracellular cGMP. Compounds of the present invention having potential pharmaceutical activity may be

selected first on the basis of their ability to modulate intracellular cGMP. Such target

compounds may be from a library of pheromone compounds to obtain compounds

having screened for desired pharmaceutical utilities which involve modulation of cGMP.

"Modulation" of intracellular cGMP may either be elevation of the cGMP level or

a decrease in cGMP level. The selection of compounds which either elevate or

decrease the level of intracellular cGMP may be carried out using various assays

known in the art. Alternatively, it is also possible, in accordance with the invention, to

separately screen for such compounds which elevate the level of intracellular cyclic

GMP using an assay in which such compounds are detected, and to separately screen

compounds which cause a decrease in intracellular cyclic GMP levels using a separate

assay. Compounds in accordance with the invention which elevate the level of

intracellular cyclic GMP will be referred to herein at times as "activators" while

compounds that decrease the level of intracellular cyclic GMP will herein be referred to

at times as "inhibitors". In addition, compounds may be selected directly which are

useful in indications known to involve cGMP.

In accordance with the present invention it has been realized that non-peptide

and non-steroid pheromones and derivative and analogs thereof are useful as a novel,

non- toxic class of compounds in the inhibition of MDR in cancer cells and in MDR-

resistant micro-organisms. Specifically, several such pheromones were shown to increase the concentration of the indicator calcein-AM, a P-gp substrate. An increase

in calcein-AM concentration is indicative of an inhibition of the outward efflux of the

calcein-AM and, thus the inhibition of P-gp activity and consequently, inhibition of MDR

in cancer cells. These results also lead to the realization that such pheromones are

useful in enhancing the efficacy of chemotherapeutic treatment of cancer and of

treatments directed against various pathogenic infections caused by microorganisms.

In accordance with the present invention that since some of the activities of

invertebrate-derived, non-peptide and non-steroid pheromones are manifested by the

inhibition of MDR in cancer cells and/or pathogenic microorganisms, such pheromones

are a source of compounds which may be used for a range of pharmaceutical uses in

anticancer and antimicrobial therapy. Compounds of the present invention, therefore,

having such potential pharmaceutical or therapeutic activity may be selected first on the

basis of their ability to inhibit MDR and thus, enhance the efficacy of chemotherapeutic

and cytotoxic treatments of cancer and microorganism-induced infection . Such target

compounds may be screened from a library of pheromone compounds to obtain

compounds having desired pharmaceutical uses which involve MDR inhibition.

Brief Description of the Drawings

Figure 1 is a schematic representation showing the level of intracellular

accumulation of calcein-AM the fluorescent P-gp substrate into P388 mouse leukemia- drug resistant cells (P388/VMDR) with increasing concentrations of the pheromone cis-

7-tetradecenyl;

Figure 2 is a schematic representation showing the effect of the pheromone cis-

11-tetradecenyl on calcein-AM accumulation into P388A MDR cells; and

Figure 3 is a schematic representation showing the effect of the pheromone 10-

dodecenyl-acetate on calcein incorporation into P388/VMDR cells.

Detailed Description of the Invention

The non-peptide, non-steroid type invertebrate-derived pheromones of the

present invention may be isolated from an invertebrate by any one of the methods

known in the art (e.g. Regnier, 1968). Alternatively, the active agent of the invention

may also be chemically synthesized. Many of the invertebrate pheromones (and most

of the insect pheromones) are synthesized either directly from or by using the fatty acid

synthesis pathway as described in, for example, Jacobson, M., U.S. Pat. No.2,900,756.

The invertebrate derived pheromones of the present invention have a molecular

weight typically less than about 500 Daltons, and more typically less than about 300

Daltons. Typically, the pheromone of the present invention comprises a straight or

branched hydrocarbon chain of variable length (typically having a length of from about

6 to 30 carbon atoms, preferably from about 7 carbon atoms to 23 carbon atoms, and

most preferably from about 9 carbon atoms to 21 carbon atoms). The hydrocarbon

chain may comprise one or more double or triple bonds which may be located at any

position in the chain, the double bonds being in the cis or trans configuration. Typically,

the side chains in a branched main hydrocarbon chain may include alkyl groups, alkenyl

groups and/or alkynyl groups, each side chain comprising from one to five carbon

atoms. The main hydrocarbon chain may also comprise or be linked to a cycloalkyl or

cycloalkenyl group having from about 3 to 7 carbon atoms.

The main hydrocarbon chain may be substituted at any location of the chain by

one or more functional groups including, for example, a hydroxyl, a ketone, an

aldehyde, an epoxy group, a carboxylic acid, an ester, a heterocyclic ring, and an

aromatic ring.

In addition, the hydrocarbon chain of the pheromone may also comprise one or

more additional groups which may, for example, be selected from ketones, halides

(such as for example, F, CI, Br, I), acetate esters, amines, thiols, short alkyls (such as

for example, methyl, ethyl, propyl, butyl, pentyl). All of the above modifications may

easily be carried out by a person versed in the art. Examples of pheromone compounds of the present invention include but are not

limited to 1 ,2-hexadecanediol, trans-5-decenyl acetate, trans-7,cis-9-dodecadienyl

acetate, cis-7-tetradecenal, trans-5-decenol, trans-8-,trans-10-dodecadienyl acetate,

trans-8-dodecenyl acetate, cis-11-dexadecenol, cis-7,cis-11-hexadecadienyl acetate,

cis-11-hexadecenal, cis-5-decenyl acetate, 4-methyl-pyrol-2-carboxylic acid methyl

ester, trans-10, trans-12-tetradecadienyl acetate, trans-11-hexadecen-1-yl acetate,

trans-11-hexadecen-l-ol, cis-3-dodecenyl-trans-2-butenoate, cis-7-tetradecenyl, cis-11-

tetradecenyl, cis-13-octadecenal, trans-8-,trans-10-dodecadienol, cis-11-hexadecenyl

acetate, cis-11-tetradecenal, 4-methyl-5-nonanol, [S]-verbenone, 3-methyl-2-

cyclohexen-1-ol, 1-methyl-butyl-2-trans-methyl-2-pentenoate, 14-methyl-8-hexadecenal,

trans-,cis-3,7-tetradecadienyl acetate, cis-7-tetradecenol, cis-7-dodecenyl acetate, cis-

6-heneicosen-11-one, cis-4-tridecenyl acetate, cis-7-tetradecenyl acetate, and cis-9-

heneicosene.

The term "effective amounf'in the context of the invention should be understood

as meaning an amount of the active agent which results in the desired therapeutic

activity including preventive effect in the treated warm-blooded animal. The effective

amount will depend on various factors such as, for example, the nature of the indication

for which the agent is used, characteristics of the treated warm-blooded animal, and

mode of administration, and will easily be determined by a person versed in the art. The effective amount of the pheromone of the present invention administered for

prevention and treatment in a warm-blooded animal, will typically be in the range of

from about 0.1 to 200 mg/kg/day, preferably from about 2.0 to 180 mg/kg/day.

In accordance with the invention pheromone analogs which comprise modified

functional groups in which, for example, an oxygen atom is replaced by a sulfur atom,

may also be used and fall within the meaning of the term "pheromone" as used herein.

Thus, for example, a hydroxyl group may be replaced by a thiol group, an ester or a

thioester. Another example of a modification may be the replacement of a hydrogen

atom by a halogen atom, such as, for example, a bromine atom. All of the above

modifications may easily be carried out by a person versed in the art.

In accordance with the invention, the derivatives and analogs of the invertebrate

pheromone substantially maintain the desired preventive or therapeutic activity of the

pheromones.

A "derivative or analog" as used herein may be any substance having the basic

structure of the invertebrate pheromone in which one of the functional groups is

modified (e.g. by one of the modifications mentioned above), but which maintains the

desired activity of the unmodified pheromone. In many cases, geometric isomers of the

invertebrate pheromone may possess a desired activity and in some cases there may be a geometrical isomer which has a higher activity than the corresponding unmodified

pheromone.

A derivative or analog which "substantially maintains" the activity of the

pheromone, means a derivative or an analog which displays a biological activity of a

magnitude which is at least 30%, preferably at least 50% of that of the pheromone. At

times, a derivative or analog which has a lower activity as compared to that of the

unmodified pheromone, may yield the same effect if its concentration is raised. As will

be appreciated, there may be derivatives or analogs which display a higher activity as

compared to the unmodified pheromone.

Where the end biological effect of the pheromone is a prevention or treatment

of a disease, condition or symptom thereof, a derivative may be regarded as

substantially maintaining this activity, if it achieves a similar preventive or therapeutic

effect at a non-toxic concentration.

It is to be understood that at times, the modification of a pheromone to form a

derivative or analog thereof may achieve desired therapeutic activity but at the same

may result in the loss or alteration of one or more inherent properties associated with

the unmodified pheromone. For use in the methods of the invention associated with the prevention and/or

treatment of diseases, conditions, and symptoms thereof, the pheromone may be

administered to a warm-blooded animal including humans by one of a variety of

administration modes, including, but not limited to oral, intravenous, intramuscular,

transdermal, subcutaneous, topical, sublingual, rectal, and nasal. In addition, the active

agent in the composition may be a volatile substance and thus has the advantage of

being administered by inhalation, resulting, in many cases in a very rapid response to

the active agent with relatively little, if any, side effects.

When the pheromone of the present invention is administered orally, it may be

administered in the form of a tablet, a pill, a capsule (e.g. a gelatin capsule), a powder,

or a pellet. Where a liquid carrier is used, the oral preparation may be in the form of a

syrup, an emulsion, or a soft gelatin capsule. Nasal administration may be by nasal

insufflation or as an aerosol, and internal administration such as rectal administration

may be by use of a suppository. For topical administration the pheromone may be, for

example, in the form of creams, ointments, lotions, solutions, gels, and transdermal

patches.

The pheromone of the present invention will typically be administered with a

pharmaceutically acceptable carrier which may be selected from a large number of

carriers known in the art and its nature will depend on the intended form of

administration and indication for which the agent is used. Tablets, pills and capsules containing the pheromone of the present invention

may also include conventional excipients such as lactose, starch, magnesium stearate.

Suppositories may include excipients such as waxes and glycerol. Injectable solutions

may comprise saline, buffering agents, dextrose, water, glycerol, ethanol and solvents

such as propylene glycol, polyethylene glycol and ethanol. Such solutions may also

comprise stabilizing agents and preservatives which are typically antimicrobial agents

(such as chlorobutol, benzyl alcohol, sodium benzoate, ascorbic acid, and phenol) and

antioxidants (such as butylated hydroxy toluene, propyl gallate, and sulfites). Enteric

coatings, flavorings, and dyes and colorants may also be used.

At times, the pheromone of the present invention may be incorporated within a

liposome prepared by any of the methods known in the art. In addition, the pheromone

may be encapsulated in inert polymerized particles such as, for example, nano

particles, microspheres, and microparticles known in the art.

The pheromones of the present invention may comprise a single active agent or

alternatively two or more such active agents which may exert a synergistic effect.

According to the method of the invention, the pheromone may be administered

separately or, alternatively, in combination with various other treatments administered

to the patient for the same or different disease, condition or symptom thereof. The pheromone of the present invention may be used to prevent and/or treat a

variety of diseases, conditions, or symptoms thereof. One such disease is cardiac

arrhythmia. Cardiac arrhythmias reflect disturbances of either impulse initiation or

impulse propagation in the heart and are caused mainly by myocardial infarction,

cardiac ischemia, aberrant conduction pathways, thyroid disease, drugs, electrolyte

disturbances, cardiomyopathy and myocarditis (Roden, O.M., 1996). In accordance

with the invention, it has been shown that the pheromone 1 ,2,-hexadecanediol has

substantive anti-arrhythmic activity.

In accordance with the present invention it has been found that non-peptide and

non-steroid pheromones, derivatives and analogs thereof are useful in the prevention

and treatment of cardiac arrhythmia. The present invention also provides a

pharmaceutical composition for the treatment of cardiac arrhythmia comprising as an

active agent an effective amount of at least one non-peptide and non-steroid

pheromone, derivative or analog thereof and a pharmaceutically acceptable carrier.

The term "cardiac arrhythmia " (referred to at times as "arrhythmia ") in the context

of the present invention encompasses any type of cardiac arrhythmia known in the art

such as those mentioned above and in Roden, D.M, 1996. The compositions and

methods of the invention are useful in the prevention and/or treatment of one or more

arrhythmia-related diseases, conditions and symptoms thereof including but not limited to a reduction in the number of VT events, a reduction in the number of VF events, and

a reduction in the number of atrial fibrillation events.

The term "prevention of arrhythmia" refers to a warm-blooded animal having a

high risk of developing arrhythmia, is treated with the composition of the invention, and

as a result does not develop an arrhythmia. The prevention of the onset of arrhythmia

may be due to the effect of the composition of the invention on one or more arrhythmia-

related parameters.

The term "treatment" refers to administration of the composition of the invention

to a warm-blooded animal suffering from arrhythmia, with the result that the arrhythmia

is reduced or cured. The improvement or curing may result from the effect of the

treatment in accordance with the invention on one or more arrhythmia-related

parameters.

The present invention further provides a pharmaceutical composition comprising

as an active agent an effective amount of at least one non-peptide and non-steroid

pheromone, derivative or analog thereof and a pharmaceutically acceptable carrier for

the prevention and/or treatment of one or more of the following: (i) ventricular

tachycardia (VT); (ii) ventricular fibrillation (VF); (iii) atrial fibrillation (AF); (iv) sinus node

disease; (v) sinus rate. The invention further provides a pharmaceutical composition comprising as an

active agent an effective amount of an invertebrate-derived non-peptide, non-steroid

pheromone having the desired therapeutic activity (i.e. modulation of cGMP levels) or

a derivative or analog of the pheromone substantially maintaining the pheromone's

activity, and a pharmaceutically acceptable carrier.

In accordance with one embodiment of the invention, the invertebrate-derived

pheromone elevates the intracellular cGMP levels by activation of GC receptor. Such

activation leads, in turn, to activation of the catalytic domain of the receptor and to

production of cGMP. Activation of particulate GC receptor was shown to be involved

in various diseases, conditions and symptoms thereof, including, for example,

peripheral vascular disease, acute congestive heart failure (CHF), renal failure and

hypertension. Thus elevation of the cyclic GMP levels by such activation may be useful

in the prevention and treatment of such diseases, conditions and symptoms thereof.

In accordance with an additional embodiment of the invention, the pheromone

may activate soluble GC receptor which elevates levels of intracellular cGMP. This

signal transduction pathway is involved, for example, in diseases, conditions, and

symptoms related to erectile dysfunction and impotence.

Furthermore, the pheromone of the invention may inhibit phosphodiesterase

(PDE) or the enzyme neutral endopeptidase (NEP). In accordance with an additional embodiment of the invention, the pheromone

decreases the level of intracellular cGMP. Such pheromones may be useful in the

prevention and treatment of diseases, conditions, and symptoms thereof such as septic

shock, neurodegenerative disorders and inflammation.

The present invention further provides a method of treating warm-blooded

animals including humans afflicted with a disease, condition, or symptoms thereof which

may benefit from modulation of cGMP levels comprising administering to the warm¬

blooded animal an effective amount of an invertebrate-derived, non-peptide, non-steroid

active agent, the active agent being selected by one or more of the screening methods

of the present invention.

The present invention also provides an invertebrate-derived, non-peptide and

non-steroid pheromone selected by one or more of the screening methods of the

present invention a pheromone having a desired therapeutic activity involving

modulation of cGMP levels.

The present invention also provides a pharmaceutical composition for the

inhibition of multi-drug resistance (MDR) in cancer cells and/or pathogenic

microorganisms, the composition comprising as an active agent an effective amount of

at least one non-peptide and non-steroid pheromone or derivative or analog thereof and

a pharmaceutically acceptable carrier. The present invention also provides a method for inhibiting MDR in the cells of

a tumor in a warm-blooded animal including humans or in microorganisms capable of

causing infection which are present in warm-blooded animals comprising administering

to the warm-blooded animal an effective amount of a non-peptide non-steroid

pheromone, derivative, or analog thereof.

The present invention provides for the use of at least one non-peptide and non-

steroid pheromone derivative or analog thereof and compositions containing the same

for inhibiting MDR in cancer cells or MDR in microorganisms.

In accordance with the invention the pheromone is useful for the treatment of

cancer, it will typically be administered in combination with an anti-cancer

chemotherapeutic treatment in order to enhance the efficacy of such treatment by

inhibiting MDR of the target cancer cells to the cytotoxic drugs associated with such

anti-cancer treatments.

Thus, in accordance with the present invention there is provided a

pharmaceutical composition comprising as an active agent an effective amount of at

least one pheromone as defined above, and a pharmaceutically acceptable carrier for

enhancing the efficacy of an anti-cancer chemotherapeutic treatment. In addition, the present invention concerns a method for enhancing the anti-

cancer effect of a chemotherapeutic treatment given to a warm-blooded animal having

cancer comprising administrating a composition comprising as an active agent an

effective amount of at least one pheromone.

Where the pheromone is used for the treatment of an infection caused by a

microorganism, the pheromone may typically be administered in combination with an

antimicrobial agent in order to enhance the efficacy of the treatment by inhibiting the

resistance of the target microorganisms. The term "microorganism" is intended in its

customary broad sense to include all microscopic organisms which are capable of

causing infection. A suitable class of microorganism is bacteria and an example of an

antimicrobial drug is an antibiotic.

Suitable examples of pheromones in accordance with the invention for use in

preventing or treating MDR of cancer cells and microorganisms include those set forth

in Table 1.

TABLE 1

Thus, in accordance with the present invention, a pharmaceutical composition

is provided comprising as an active agent an effective amount of at least one non-

peptide and non-steroid pheromone or derivative or analog thereof and a

pharmaceutically acceptable carrier for enhancing the efficacy of an antimicrobial drug

in the treatment of microorganism-induced infection.

In addition, the present invention provides a method for enhancing the

antimicrobial effect of a cytotoxic treatment given to a warm-blooded animal having a

microorganism-induced infection comprising administering a composition comprising

pheromone, derivative or analog thereof to the warm-blooded animal.

The pheromones of the present invention may also be used for the prevention

and treatment of acute congestive heart failure (CHF) which is among the leading causes of hospitalization in the U.S. The most common cause of acute congestive

heart failure is coronary artery disease, i.e. narrowing of the arteries which supply blood

to the heart muscle. CHF is also characterized by the build-up of body fluid in the lungs

and elsewhere. Typically diuretics and vasodilators (e.g. angiotensin converting

enzyme (ACE) inhibitors) are currently used for the treatment of CHF by i.v. infusion

(which often requires hospitalization of the treated individual).

Additional examples of diseases, conditions, and symptoms thereof which may

be prevented and/ortreated in accordance with the invention, are the prevention and/or

treatment of vascular and hemorheological diseases. Rigidity of the membrane of red

blood cells (RBCs) or narrowing of the capillary's lumen leading to impaired passage

of RBCs through these small vessels characterizes a variety of inherited diseases as

well as various acquired pathological states such as thalassemia, hemolytic anemias,

heart failure, and stroke. Therapeutics used today for treating peripheral vascular

disease include, for example pentoxifylline (Trental™, Hoechst Marion Roussel). In

accordance with the present invention, it has been shown that invertebrate-derived,

non-peptide and non-steroid pheromones have an effect on the structural

hemorheological parameters of RBCs.

The pheromones of the present invention may, for example, also be used in the

field of fertility. The non-steroidal nature of the active agent of the invention reduces

the adverse side effects associated with commonly used steroids for this purpose. The pheromone of the present invention may further be used for the prevention

or treatment of impotence, such as, for example, by preventing or treating erectile

dysfunction. For this utility (as well as for others where a rapid response is desired) it

is preferred that the active agent is a volatile invertebrate pheromone. The composition

containing the active agent may then be administered by inhalation, resulting in a very

rapid effect.

Additional examples of indications for which the pheromone of the present

invention may be used, include a female contraceptive, a treatment of diabetes, an

erythropoietin mimetic compound, a treatment of neurological disorders, an analgesic

agent, and an anti-anxiety agent.

The above embodiments should not be construed as limiting and the

pheromones of the present invention may be used for the prevention and treatment of

a variety of additional conditions which are not specifically mentioned herein.

The pheromones of the present invention may be tested for a variety of

diseases, conditions, symptoms in the following manner.

Using the Bactec system (Roberts G.D. et al., 1983), bacteria strains from solid

or fluid media are tested for sensitivity to antibiotic drugs with and without prior addition

of tested pheromones of the present invention. One drug-free control with undiluted, and one drug-free control with diluted (1 :100) bacterial suspension are used. Vials

containing tested samples of resistant bacteria, with or without antibiotic drug and with

or without tested pheromone are used. The vials are incubated at 36°C for 4 days.

The daily recordings of bacteria viability are read. A decrease in the viability of the

bacteria in the presence of the tested pheromone indicates an effect of the tested

pheromones of the invention on inhibiting bacteria resistance to the given antibiotics,

i.e. indicates an MDR-inhibiting activity of the pheromone on the bacterial cells.

For anti-diabetic activity, the tested pheromone is incubated with cells expressing

insulin receptors on their plasma membranes' outer leaflet such as primary adipocytes.

The binding of the tested pheromone to the insulin receptor is determined by any of the

methods known in the art. In addition, in a radiolabeled glucose transport assay, the

effect of pheromones on receptor-mediated glucose uptake is determined in these cells.

For anti-non-insulin-dependent diabetes mellitus (NIDDM) activity, the effect of

pheromones on insulin blood level is tested in experimental animals using a routine

radio-immuno assay (RIA)fordetermining insulin concentrations in blood samples. The

insulin concentration as well as glucose level is determined in the blood of animals

receiving injections of the tested pheromone and compared to the level of glucose in

animals injected with a control preparation without the tested pheromone.

In addition, the effect of the tested pheromone is tested in vitro using the lipolysis assay. Cultured adipocytes are incubated with the tested pheromone and the level of

lipolysis in the cells is compared to the level in cells which are not incubated with the

tested pheromone (negative control) as well as in cells incubated with insulin which inhibits lipolysis (positive control).

For testing pheromones for erythropoietin (EPO) mimetic activity, cell

proliferation of cells which are dependent on EPO is determined in the presence of the

tested pheromone. Ba/F3 cells are a pro-B cell line lacking endogenous EPO

receptors. Transfection of these cells with EPO-R cDNA makes them dependent on

EPO for proliferation. The tested pheromone is incubated with Ba/F3 cells and their

level of proliferation is determined by methods known in the art.

Binding of the pheromones to the EPO-receptor is tested by competitive receptor

binding assay in which their ability to inhibit binding of ¹²⁵l-labeled EPO to the EPO-R

is determined.

In addition to the above mentioned uses of the active agent of the invention in

vivo, pheromones may also be used ex vivo (or in vitro). For example, red blood cells

may be obtained from a patient suffering from vascular disease and incubated ex vivo

with the pheromones of the present invention to increase the deformability of the

membranes of the red blood cells. The treated cells may then be re-administered to the

treated patient. In accordance with one aspect of the invention for a composition and treatment

of arrhythmia, the pheromone is a diol, i.e. a hydrocarbon chain as described above

having two hydroxyl groups. By a preferred embodiment, the diol is a 1 ,2-diol, some

non-limiting examples being 1 ,2-hexadecanediol, 1 ,2, octadecanediol, 15-Methyl-1 ,2-

hexadecanediol.

Some examples of pheromones useful in the present invention for elevating or

lowering cGMP levels include trans-8-dodecenyl acetate, cis-11-hexadecenol, cis-11-

hexadecenyl acetate, cis-7-tetradecenal, cis-13-octadecenal, and trans-,trans-8,10-

dodecadienol which may be used with the principles of the present invention and which

were found in accordance with the screening methods of the present invention to have

cGMP level modulating activity.

Where the pheromones are first screened on the basis of their ability to modulate

intracellular cGMP levels, the effective amount will either elevate or decrease

intracellular cGMP levels as determined by a suitable assay. An effective amount of

the pheromone is typically an amount giving rise to at least 30% increase or decrease

in level of intracellular cGMP as compared to the intracellular level of cGMP in the same

cells, which are not stimulated by the pheromone of the present invention. The

methods for determining the level of intracellular cGMP include, for example, any

available commercial radioimmuno-assay kits for measuring cGMP or by degradation

of cGMP with cyclic nucleotide phosphodiestherase (Ziegelsberger, G. et al., 1990). An additional example is an assay based on use of SV40 transformed cat iris sphincter

smooth muscle cells (SV-CISM-2) (Ding, K.H. et al., 1999). These cells have an

increased sensitivity to compounds which modulate intracellular cGMP levels. It is to

be noted that screening of a compound for its ability to modulate cGMP levels may be

carried out using more than one type of assay and on various types of cells.

The pharmaceutical composition of the invention may be used to prevent and/or

treat a variety of diseases, conditions, and symptoms thereof. Where the

pharmaceutical composition is capable of activating particulate GC receptor, the

composition may, for example, be useful in the prevention or treatment of vascular and

hemorheological diseases. Rigidity of the membrane of RBCs or narrowing of the

capillary's lumen leading to impaired passage of RBCs through these small vessels

characterizes a variety of inherited diseases as well as various acquired pathological

states such as thalassemia, hemolytic anemias, heart failure, stroke, and the like.

Therapeutics used today for treating peripheral vascular disease include, for example

pentoxifylline (Trental™, Hoechst Marion Roussel).

By way of example, the effect of pheromones on guanylyl cyclase (GC) activity

and the cGMP levels in RBCs may be incubated in the presence and absence of

pheromones of the invention and the level of GC activity in the RBCs evaluated

according to the method of Ishii et al., (1989). In addition, RBCs derived from patients

suffering from various diseases, conditions, and symptoms thereof, may be incubated in the presence and absence of the pheromones and the effect of the pheromones on

the cells may be tested using the methods described in Ishii et al. (1989) and compared

to the effect of the pheromones on RBCs obtained from healthy patients.

The pharmaceutical composition of the invention may also be used for the

prevention and treatment of acute congestive heart failure (CHF) which is among the

leading causes of hospitalization in the U.S. The most common cause of acute

congestive heart failure is coronary artery disease, i.e. narrowing of the arteries which

supply blood to the heart muscle. CHF is also characterized by the build-up of body

fluid in the lungs and elsewhere. Typically diuretics and vasodilators (e.g. angiotension

converting enzyme (ACE) inhibitors) are currently used for the treatment of CHF by i.v.

infusion (which often requires hospitalization of the treated individual).

Where the composition of the invention is capable of activating soluble GC

receptor, it may, for example, be used in the field of fertility. It has now been shown

that humans are sensitive to pheromones and that there is a high degree of

conservation in both pheromones and receptors across species. Thus, it has been

realized in accordance with the present invention that invertebrate pheromones may be

useful in fertility treatments in warm blooded animals including humans. The non-

steroidal nature of the active agent of the invention reduces the adverse side effects

associated with commonly used steroids. The pharmaceutical composition of the invention may also be used for the

prevention or treatment of impotence. Modulation of cyclic GMP levels have been

shown in some cases to alleviate erectile dysfunction. In accordance with the invention,

a composition comprising an active agent which inhibits PDE is provided for the

prevention and treatment of erectile dysfunction. By a preferred embodiment, the active

agent is a volatile invertebrate pheromone, which provides for a composition that may

be administered by inhalation, resulting in very rapid elevation of cGMP levels in the

target cells. The pharmaceutical composition of the invention may further be used for

the treatment of neurological disorders, as an analgesic, or as an anti-anxiety agent.

The pheromone containing pharmaceutical compositions of the present invention which

decrease the level of intracellular cyclic GMP, may be used for the treatment of

diseases, conditions, and symptoms thereof associated with high levels of intracellular

cyclic GMP such as, for example, septic shock, neurodegenerative disorders and

inflammation. These conditions typically involve excessive production of nitric oxide

(NO). (Hobbs, A.J. et al., 1999). Most of the pathological effects of NO are caused by

its activation of the soluble guanylate cyclase enzyme which in turn results in the

elevation of intracellular cGMP. Therefore, pharmaceutical compositions of the

invention comprising pheromones which are capable of decreasing the level of cGMP

may have an effective therapeutical effect on such conditions.

The foregoing discussion discloses and describes merely exemplary

embodiments of the present invention. One skilled in the art will readily recognize from such discussion, from the examples, and from the claims, that various changes,

modifications and variations can be made therein without departing from the spirit and

scope of the invention as encompassed by the claims forming part of the application.

The following examples are submitted for illustrative purposes only and are not

intended to limit the invention as encompassed by the claims forming part of the

application.

EXAMPLE 1

Anti-hemolytic Activity of Pheromones of the Invention

Non-peptide, non-steroid pheromones derived from insects in the concentrations

as shown in Table 2, were tested by a simple hemolytic assay on red blood cells

(RBCs) (Horikawa, K., 1997). In the assay, approximately 50 percent of red blood cells

are protected against hemolysis at a concentration of 0.425% NaCI. Samples of RBCs

are tested and observed with and without the presence of the tested pheromone. The

anti-hemolytic effect of the tested pheromone is determined by the percent of protection

of the affected RBCs from hemolysis obtained in the presence of the tested pheromone

as compared to the percent hemolysis of the cells in NaCI only. The anti-hemolytic

effect of pentoxifylline (trental) was also tested.

As shown for example in Table 2, there was a 28.5% decrease in the number of

RBCs affected when using trans-5-decenyl acetate. As also shown in Table 2, the

results of the above experiment showed that about 20% of the tested pheromones

exhibited anti-hemolytic activity at a concentration of lO^M and about 5% of the

pheromones tested exhibited such activity at concentrations as low as 10^"10M.

Such low effective concentrations are characteristic of receptor mediated

phenomena and thus suggest that the pheromones's anti-hemolytic effect was

apparently through an increase in the deformability of the RBCs. Hemolysis of RBCs obtained at 0.425% NaCI was used as a reference. The

numbers represent % protection against hemolysis of RBCs in the presence of a tested

pheromone of the present invention at 0.425% NaCI.

Table 2

Results indicating Anti-hemolvtic effect of pheromones

on human RBCs in vitro

indicates less than 15% reduction in hemolysis

EXAMPLE 2

Testing for the effect of pheromones on cellular deformability of RBC by

laser-assisted optical rotational cell analyzer

The effect of the tested pheromones on cellular deformability of RBCs was

determined by a laser-assisted optical rotational cell analyzer (L.O.R.C.A) in

accordance with Hardemann et al. (Hardemann, M.R. et al., 1994). In this method,

RBCs were subjected to laminar sheer stress and their capacity to deform under stress

was determined. The resulting change in cell shape was continuously monitored by

laser defractommetry detected by a video camera and an elongation index (El) was

calculated on the basis of the extent of the change in the shape of the RBCs. An effect

of the tested pheromone on the deformability of RBCs results in a higher El. The El

was determined by applying different stresses on the RBCs, and the results, presented

in Table 3 below, are shown at 3 pascal units (Pa) and at 30 Pa, respectively.

The effect of 20 pheromones of the invention at a concentration of 10^"4 M was

tested on blood obtained from three different donors:

1 ) A healthy 59 year old man;

2) A healthy 54 year old man; and

3) A healthy 25 year old man.

The measurement of the RBC deformability was carried out according to the protocol of Hardemann et al., (1994). Statistical analysis was performed using the

known Students' t-test.

The results between 3 pascals and 30 pascals obtained from donor 3 (the 25

year old man) were very similar to each other. There was no statistically significant

difference in the results between the presence and absence of the pheromones at 3

and 30 pascals, respectively. Accordingly, the pheromones had no effect on the

deformability of the red blood cells of donor 3.

The effective concentration of the pheromones was 10^M and while some of the

pheromones including E-5-decenyl acetate, 4-methyl-pyrol-2-carboxylic acid-

methylester, and E-8-dodecenyl-acetate, had an effect on the deformability of the red

blood cells of both other donors, others including cis-7-tetradecenyl, E-E-10,12-

tetradecadienyl acetate, E-11 -Hexadecen-1 -ol, and Z-3-dodecenyl-E-2-butenoate, were

effective in one donor only.

The effect of six of the above tested pheromones was determined in an anti-

hemolytic assay as well (as described above). As seen in Table 2, all of these six

tested pheromones had an effect on the deformability of the red blood cells in this

assay. Table 3

* Compounds which were found to be effective in both donors at 3 Pascal. The initials "n.d." mean not determinable.

EXAMPLE 3

Anti-arrhvthmic activity of pheromones in coronary ligation.

reperfusion arrvthmia in anesthetized rats

The anti-arrhythmic activity of the pheromone 1 ,2,hexadecanediol was measured

using the occlusion-reperfusion model in rats.

Materials and Method:

1 ,2-hexadecanediol was obtained from Sigma-Aldrich (Sigma, Israel). The diol

may be prepared by methods known in the art (see, for example, Jacobson et al., or

Nakamura et al, 1993). The vehicle used when administering the 1 ,2-hexadecanediol

contained 0.5% ethanol, 0.4% Tween 80, 0.75% Lipoid and water up to 100%.

Experimental Assay:

The anti-arrthymic effect of 1 ,2-hexadecanediol was tested in an occlusion-

reperfusion induced arrhythmia model in anesthetized rats. Following occlusion of the

left main coronary artery, very marked ventricular dysrhythmias occurred. This model

is acknowledged as suitable for testing drugs with potential anti-arrhythmic activities

(see, Szekers, et al., 1975). 17 male rats were anesthetized by intraperitoneal injection of 60 mg/kg

pentobarbital sodium. The trachea was intubated to allow artificial ventilation using a

Starling pump. A catheter was placed in an internal jugular vein for administration of

the test pheromone compounds.

Monitoring Physiological Parameters:

Peripheral blood pressure and ECG lead II were recorded continuously during

the entire experiment. Rectal temperature was maintained at 38°C. The chest was

opened by left thoracotomy at the fourth intercostal space. After opening the

pericardium, the heart was exteriorized by placing gentle pressure on the chest walls

and using a thin silk thread (Ethicon 1.5 metric, 5-0) attached to an atraumatic needle

which was placed around the left coronary artery about 2-3 mm distal of the origin of

the left coronary artery for later ligation. The animal was then ventilated with room air

using a stroke volume of 1 ml/100 g of body weight at a rate of 54-56 stroke/min. The

heart was then placed back in the chest cavity. It is noted that any animal which

experienced dysrhythmias or a sustained fall in mean arterial blood pressure to less

than 70 mm Hg in response to the above-described preparatory procedure, was

discarded from the study.

After an equilibration time of approximately 45 minutes, 1 ,2-hexadecanediol or the vehicle (control) was administered by intravenous (i.v.) injection, and 10 minutes

thereafter, the ligature at the left coronary artery was occluded for a total duration of 10

minutes then subsequently reperfused for 10 minutes. For i.v. injection, the test

compounds were dissolved or suspended in the vehicle 30 minutes before occlusion.

Measurements and Evaluation:

Evaluation of anti-arrhythmic and/or anti-fibrilatoric activities was carried out

basically according to Lambeth conventions (Walker, M.J.A., et al., 1988).

During the occlusion period, the number of rats with ventricular tachycardia (VT)

were determined. VT is defined herein as any run of seven or more consecutive

ventricular extrasystoles (premature ventricular contractions - PVC).

During the reperfusion period, the following parameters were measured in each

rat:

i. the number of ventricular trachycardia (VT) events (as defined above);

ii. the number of ventricular fibrillation (VF) events;

iii. sinus rate recovery - the number of seconds measured until sinus rate

recovery in the rat; and

iv. onset of arrhythmia. The above parameters were evaluated separately and compared with a control

group comprised of rats which received an i.v. injection of the vehicle.

Changes of parameters in the rats treated with 1 ,2-hexadecanediol were

compared to the parameters measured in rats receiving the control vehicle. The

statistical significance of the measured parameters was assessed by Student's t-test

known to those in the art.

The total amount of 1 ,2-hexadecanediol or the control vehicle which were

infused during the 20 minute infusion period was based on an amount of 1 ml for a 250

g rat, corresponding to a volume dosage of 4 ml/kg.

The solutions comprising either the 1 ,2-hexadecanediol or the control vehicle

were prepared at concentrations appropriate for selected dose levels and infusion

volume dosage of 4 ml/kg/20 minute for a total dose rate at about 2 mg/kg.

Results:

As seen in Table 4A, most of the rats (88%) which were administered with the

control vehicle, showed VT events. A statistically significant smaller number of the rats

(22%) which were administered with 1 ,2-hexadecanediol, showed VT events. Table 4A

Anti-arrhvthmic activity of 1 ,2-hexadecanediol in Coronary Artery

Ligation Arrhythmia in Anesthetized Rats

Occlusion Period

As seen in Table 4B, the rats receiving 1 ,2-hexadecanediol had a stastically

significant lower number of VT and VF events as compared to the rats receiving the

control vehicle. The 1 ,2-hexadecanediol receiving rats also had a faster sinus rate

recovery than that of the control rats.

Table 4B

Anti-arrhvthmic activity of 1 ,2-hexadecanediol in Coronary Artery

Reperfusion Arrhythmia in Anesthetized Rats

Reperfusion Period

The above results clearly show a substantive anti-arrhythmic activity of the

compound 1 ,2-hexadecanediol in anesthetized rats as compared to the control group.

EXAMPLE 4

Effect of tested pheromones on Multi-Drug Resistance

in mouse leukemia cells

Materials and Methods:

Fetal calf serum (FCS) and all tissue culture reagents were purchased from

Biological Industries (Beth Haemek, Israel). Calcein-Am and Verapamil, were

purchased from Sigma (St. Louis, MO).

Pheromones

The three pheromones that were tested were cis-7-tetradecenyl; cis-11-

tetradecenyl; and trans-10-dodecenyl acetate.

Cell culture

P388 wild type, and P388 mouse leukemia-drug resistant (P388/VMDR) cells

were grown in suspension in RPMI-1640 medium together with 10% fetal calf serum. Drug Uptake (transport) Assay (Calcein-AM uptake) - The Protocol

MDR was assayed by measuring P-gp-mediated extrusion of the fluorescent dye,

Calcein-AM out from cells. Calcein-AM serves as a substrate P-gp and is extruded

from MDR cells 10-1000-fold more efficiently in comparison to non-MDR cells. The

effectiveness of various pheromones of the invention in preventing or inhibiting MDR

was tested in the following manner.

Drug-resistant P388 cells growing in suspension were used for the drug uptake

assay. Cells were grown overnight in a culture medium, washed twice and

resuspended in PBS. Approximately 200,000 cells were placed in each well of 96-well

plastic dish in 100 μl of PBS medium. The dish containing the thus prepared cells was

placed with the tested pheromone at the desired concentrations, in triplicates, and left

to equilibrate for 10 minutes. Calcein-AM to 250 nM was added and entered the cells

where it is hydrolyzed to the non-permeable fluorescent Calcein form, which

accumulates within the cell. The progress of the transport reaction was followed on the

fluorescent plate reader for 60 minutes at 37°C. Fluoresence was read with the

excitation wavelength set at 485 nm, emission at 538 nm. The dye uptake curve was

fitted to an exponential curve and the true initial rate was calculated. Cvtotoxicitv Assay (MTT) Methodology:

Cell resistance to chemotherapy was determined in different drug-sensitive and

drug-resistant cell lines by using the MTT cell proliferation/survival assay. The MTT

assay is based on the conversion of 3(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium

bromide to formazan by living cells. EC50 values for cell killing by cytotoxic drug, in the

presence or absence of the tested pheromone provided a quantitative tool for assessing

the effectiveness of the pheromone in reversing (inhibiting) MDR in cells.

Results:

As shown in Figures 1-3, the three tested pheromones cis-7-tetradecenyl, cis-11-

tetradecenyl and trans-10-dodecenyl acetate showed MDR inhibitory activity to

concentrations up to 50 μM as shown from a base value of between 0.2 and 0.4 up to

about 0.9. The effective concentration of cis-7-tetradecenyl and cis-11 -tetradecenyl

was between 10 to 50 μM while the effect of trans-10-dodecenyl acetate was found to

be dose-dependent with IC₅₀ of 1-2 μM. The saturation curve of trans-10-dodecenyl

acetate resembled the saturation curve typically seen using the classical MDR-inhibitor

verapamil.

In summary, the above results show substantive MDR-inhibiting activity of three

tested pheromones of the invention on leukemic cells. EXAMPLE 5

Effect on cGMP formation in SV-CISM cells

The effects of several tested pheromones on cGMP production was determined

using cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells

as described in Ding, K.H. et al., (1999). As seen in Table 5A below, several of the

tested pheromones (activators) caused an increase in the level of intracellular cGMP

production in SV-CISM cells, as compared to the level of cGMP formation in the these

cells without the presence of the tested pheromones.

Table 5A

Activator Pheromones effect on cGMP formation in SV-CISM cells

As seen in Tables 5B and 5C, several other tested pheromones ("inhibitors")

caused a decrease in the level of intracellular cGMP in SV-CISM cells as compared to

the level of cGMP formation in these cells without the presence of the tested

pheromone.

Table 5B

Inhibitor Pheromones effect on cGMP formation in SV-CISM cells

Table 5C

Inhibitor Pheromones effect on cGMP formation in V- ISM cell

EXAMPLE 6

Formulation Containing Pheromone Compounds of the Present Invention

Suitable for Parenteral Administration

4.5 grams (g)of beta-hydroxypropylcyclodextrin (beta-HPCD) were added to 5.5

g of purified water and mixed together until the beta-HPCD was completely dissolved

and a clear viscous solution was formed. 5 mg of a pheromone compound (e.g. 1 ,2-

hexadecanediol) of the present invention was added to the prepared HPCD solution

and mixed under heating at 65-70X until the pheromone compound was completely

dissolved. The pheromone solution was passed through a nylon membrane filter having a pore size of about 0.45 micron. The filtered solution was then allowed to cool.

The prepared solution can be diluted to a desired concentration using 45% water

solution of HPCD.

EXAMPLE 7

Formulation Containing Pheromone Compounds of the Present Invention

Suitable for Parenteral Administration

50 mg of hexadecanol was melted with 950 mg of Tween-80 in a water bath.

The composition was then allowed to cool. Upon cooling, the gel-like composition was

dissolved and an amount of a pheromone compound (e.g. 1 ,2-hexadecanediol) of the

present invention was dissolved into 99 ml of warm purified water. A transparent

micellar solution was thus formed suitable for parenteral administration.

EXAMPLE 8

Formulation Containing Pheromone Compounds of the Present Invention

Suitable for Parenteral Administration

A microemulsion composition was prepared by mixing 1800 mg of Tween-80, a

non-ionic surfactant with 1500 mg of medium chain triglycerides oil (CRODAMOL™

TGCC). 100 mg of a pheromone compound (e.g. 1 ,2-hexadecanediol) of the present

invention was dissolved into the surfactant-oil mixture under slight heating at 45-50°C. Optionally, 200 mg of a cosolvent such as enthoxydiglycol (TRANSCUTOL™,

Gattefosse, France) may be added for improved stability. The resulting solution was

filtered through a 0.22 micron membrane filter for sterility and the resulting solution was

suitable for parenteral injection.

EXAMPLE 9

Formulation Containing Pheromone Compounds of the Present Invention

Suitable for Parenteral Administration

A microemulsion composition was prepared by mixing 3600 mg of Tween-80, a

non-ionic surfactant with 2700 mg of medium chain triglycerides oil (CRODAMOL™

TGCC). 100 mg of a pheromone compound (e.g. 1 ,2-hexadecanediol) of the present

invention was dissolved into the surfactant-oil mixture under slight heating at45-50°C.

Upon cooling, 300 mg glycol laurate (LABRASOL™) was added to the final solution and

mixed thoroughly until dissolved. The solution was then filtered through a 0.22 micron

membrane filter for sterility and the resulting solution was suitable for parenteral

injection.

EXAMPLE 10

Formulation Containing Pheromone Compounds of the Present Invention

Suitable for Oral Administration

400 mg of a pheromone compound (e.g. 1 ,2-hexadecanediol) of the present

invention was admixed with 1.0 g of polyvinylpyrrolidone (PVP K-25™, BASF). The

admixture was dissolved in 20 ml of ethyl alcohol USP grade. The resulting solution

was used for granulation of a mixture of 4.6 g of dibasic calcium phosphate dihydrate

(EMCOPRESS™, Mendell Co.) and 5.9 g of microcrystalline cellulose (AVICEL®

pH102, FMC). The resulting granulation was dried at 45°C and passed through a

stainless steel sieve (#16 mesh). The sieved granules were mixed with 0.1 g of

magnesium stearate. 900 mg of the final granulation was encapsulated in a size 00

hard gelatin capsule, providing 30 mg of the pheromone compound per capsule.

EXAMPLE 11

Formulation Containing Pheromone Compounds of the Present Invention

Suitable for Oral Administration

400 mg of a pheromone compound (e.g. 1 ,2-hexadecanediol) of the present

invention was admixed with 2.0 g of isopropyl palmitate, 4 g of MIRJ®-52 (PEG-40

stearate, USP/NF grade), and 1.0 g of polyvinylpyrrolidone (PVP K-25™, BASF). The

admixture was dissolved in 30 ml of ethyl alcohol USP grade. The resulting solution was used for granulation of a mixture of 5.6 g of calcium silicate (HIPERSORB™,

Daminco) and 1.9 g of dibasic calcium phosphate dihydrate (EMCOPRESS™, Mendell

Co.). The resulting granulation was dried at 45°C and passed through a stainless steel

sieve (#16 mesh). The sieved granules were mixed with 0.1 g of magnesium stearate.

750 mg of the final granulation was encapsulated in a size 00 hard gelatin capsule,

providing 20 mg of the pheromone compound per capsule.

EXAMPLE 12

Formulation Containing Pheromone Compounds of the Present Invention

Suitable for Suppository Use

125 mg of a pheromone compound (e.g. 1 ,2-hexadecanediol) of the present

invention was melted with 3.6 g of a suppository base comprising cacao butter (99.6%)

and butylated hydroxytoluene (0.4%). The melted composition was mixed well with a

spatula and then molded into four oval suppositoria using a suitable mold. Weighing

960 mg each, with suppositoria containing about 30 mg of the pheromone compound.

EXAMPLE 13

Formulation Containing Pheromone Compounds of the Present Invention

Suitable for Topical Administration

An ointment comprising 5% a pheromone compound (e.g. 1 ,2-hexadecanediol) of the present invention was prepared by mixing 500 mg of pheromone compounds with

7.5 g of white petrolatum, 0.5 g lanolin and 1.5 g of POLA WAX™ (Emulsifying wax NF

grade, Croda). The resulting mixture was further mixed until cooling which resulted in

an oleaginous ointment.

EXAMPLE 14

Formulation Containing Pheromone Compounds of the Present Invention

Suitable for Topical Administration

A water-in-oil emulsion cream containing 1% a pheromone compound (e.g. 1 ,2-

hexadecanediol) of the present invention was prepared by dissolving 1 g of pheromone

compounds in a mixture of 20 g of isopropyl palmitate and 10 g of

POLAWAX™(Emulsifying wax NF grade, Croda) at 70°C to form a lipid phase. A water

phase comprising 66 g of purified water, 2.5 g of glycerin and 0.5 g of phenoxyethanol

was heated to 70°C, then slowly added to the lipid phase, and vigorously mixed while

cooling. Upon cooling, a stable cream was obtained and packaged in an airtight

container.

EXAMPLE 15

Formulation Containing Pheromone Compounds of the Present Invention

Suitable for Topical Administration

A clear gel preparation containing 0.5% a pheromone compound (e.g. 1 ,2-

hexadecanediol) of the present invention was prepared by slowly adding 2.5 g of

hydroxypropylcellulose (Klucel HF) to 37.0 g of purified water. The solution was heated

to 70°C. The heated solution was mixed while allowing the temperature to fall to room

temperature resulting in a viscous gel as a water phase. Separately, 0.5 g of a

pheromone compound (e.g. 1 ,2-hexadecanediol) of the present invention was dissolved

in a mixture of 40 g ethoxydiglycol (TRANSCUTOL™), 10 g of isopropyl myristate, and

10 g of PLURONIC™ F-127 to form an organic phase. The water and organic phases

were combined and slowly mixed until a uniform composition was obtained. After

degassing for 24 hours, a smooth clear gel was formed.

Claims

What is Claimed is:

1. A method of screening for compounds having a desired therapeutic

activity, comprising the steps of:

(a) providing a biological model for screening of the compounds, said

model being predictive of the desired therapeutic activity;

(b) testing compounds from a library of compounds in said model, said

library comprising at least one pheromone compound selected from invertebrate

derived, non-peptide and non-steroid pheromones, derivatives or analogs thereof; and

(c) selecting at least one of said compounds showing the desired

therapeutic activity in said model.

2. A method of selecting compounds having a desired therapeutic activity,

comprising the steps of:

(a) providing data relating to the three-dimensional (3D) structure of

at least a pharmacophore of a compound known to have the desired therapeutic activity;

(b) providing a library of compounds, said library comprising at least

one pheromone compound selected from invertebrate-derived, non-peptide and non-

steroid pheromones, derivatives, or analogs thereof; and

(c) analyzing the 3D structure of one or more compounds of said

library and selecting a compound having a domain with a 3D structure identical or similar to the 3D structure of said pharmacophore.

3. The method of claim 2 further comprising testing the selected compound

in a relevant biological model which is predictive of said desired therapeutic activity.

4. The method of claim 1 wherein the desired therapeutic activity is the

inhibition of MDR in cancer cells, tumors and microorganisms.

5. The method of claim 1 wherein the desired therapeutic activity is the

modulation of cGMP levels.

6. The method of claim 1 wherein the desired therapeutic activity is anti-

arrhythmic activity.

7. The method of claim 1 wherein the at least one pheromone compound

has a molecular weight of less than about 500 Daltons.

8. The method of claim 1 wherein the pheromone compound has a straight

or branched saturated or unsaturated hydrocarbon chain having from about 6 to 30

carbon atoms.

9. The method of claim 8 wherein the hydrocarbon chain is substituted with at least one member selected from the group consisting of an alkyl group, an alkenyl

group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group, a hydroxyl, a halide,

an amine, a thiol, a thioester, a ketone, an aldehyde, an epoxy group, a carboxylic acid

group, as ester, an aromatic group, and a heterocyclic group.

10. The method of claim 1 wherein the pheromone compound is selected from

the group consisting of 1 ,2-hexadecanediol, trans-5-decenyl acetate, trans-7,cis-9-

dodecadienyl acetate, cis-7-tetradecenal, trans-5-decenol, trans-8-,trans-10-

dodecadienyl acetate, trans-8-dodecenyl acetate, cis-11-dexadecenol, cis-7,cis-11-

hexadecadienyl acetate, cis-11-hexadecenal, cis-5-decenyl acetate, 4-methyl-pyrol-2-

carboxylic acid methyl ester, trans-10, trans-12-tetradecadienyl acetate, trans-11-

hexadecen-1 -yl acetate, trans-11 -hexadecen-1 -ol, cis-3-dodecenyl-trans-2-butenoate,

cis-7-tetradecenyl, cis-11 -tetradecenyl, cis-13-octadecenal, trans-8-,trans-10-

dodecadienol, cis-11-hexadecenyl acetate, cis-11-tetradecenal, 4-methyl-5-nonanol,

[S]-verbenone, 3-methyl-2-cyclohexen-1 -ol, 1 -methyl-butyl-2-trans-methyl-2-

pentenoate, 14-methyl-8-hexadecenal, trans-,cis-3,7-tetradecadienyl acetate, cis-7-

tetradecenol, cis-7-dodecenyl acetate, cis-6-heneicosen-11-one, cis-4-tridecenyl

acetate, cis-7-tetradecenyl acetate, and cis-9-heneicosene.

11. The method of claim 9 wherein the pheromone compound is a diol.

12. The method of claim 11 wherein the diol is a 1 ,2-diol.

13. The method of claim 1 wherein the pheromone compound is selected from

the group consisting of 1 ,2-hexadecanediol, 1 ,2-octadecanediol, and 15-methyl-1 ,2-

hexadecanediol.

14. The method of claim 1 wherein the pheromone compound is selected from

the group consisting of cis-7-tetradecenyl, cis-11 tetradecenyl, trans-10-dodecenyl

acetate, pentyl nonanoate,3,5-decadienyl acetate, 11 , 11-difluoro-9-dodecenyl acetate,

9-tetradecenyl nitrate, 13-hexadecen-11-ynal, 9,12-octadecadienal, 3,9-6,7-

epoxydocosadiene, heptacosane, toluquinone, dodecalactone, quinazolinone,

cyclopentyl ketones, alkylpyrazines, indolizine, 9-oxo-2-decenoicacid, pyrrolizidine, and

castoramine.

15. The method of claim 1 wherein the pheromone compound is selected from

the group consisting of trans-3-tridecenyl acetate, 1 ,7-dioxaspiro-(5-5) undecane, cis-7-

tetradecenal, trans-8-dodecenyl acetate, cis-11-hexadecenol, cis-13-octadecenal,

trans-8-,trans-10-dodecadienol, cis-11-hexadecenyl acetate, 4-methyl-5-nonanol, [S]-

verbenone, 3-methyl-2-cyclohexen-1-ol, 1-methyl-butyl-2-trans-methyl-2-pentenoate,

and 14-methyl-8-hexadecenal.

16. A pharmaceutical composition comprising an effective amount of a

selected compound obtained by the method of claim 1 in combination with a

pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 16 wherein the effective amount

is from about 0.1 to 200 mg/kg/day.

18. The pharmaceutical composition of claim 17 wherein the effective amount

is from about 2.0 to 180 mg/kg/day.

19. The pharmaceutical composition of claim 16 wherein the pheromone

compound is selected from the group consisting of 1 ,2-hexadecanediol, trans-5-decenyl

acetate, trans-7,cis-9-dodecadienyl acetate, cis-7-tetradecenal, trans-5-decenol, trans-

8-,trans-10-dodecadienyl acetate, trans-8-dodecenyl acetate, cis-11-dexadecenol, cis-

7,cis-11 -hexadecadienyl acetate, cis-11 -hexadecenal, cis-5-decenyl acetate, 4-methyl-

pyrol-2-carboxylic acid methyl ester, trans-10, trans-12-tetradecadienyl acetate, trans-

11 -hexadecen-1 -yl acetate, trans-11 -hexadecen-1 -ol, cis-3-dodecenyl-trans-2-

butenoate, cis-7-tetradecenyl, cis-11 -tetradecenyl, cis-13-octadecenal, trans-8-,trans-

10-dodecadienol, cis-11 -hexadecenyl acetate, cis-11-tetradecenal,4-methyl-5-nonanol,

[S]-verbenone, 3-methyl-2-cyclohexen-1 -ol, 1 -methyl-butyl-2-trans-methyl-2-

acetate, cis-7-tetradecenyl acetate, and cis-9-heneicosene.

20. The pharmaceutical composition of claim 16 wherein the pheromone

compound is selected from the group consisting of trans-3-tridecenyl acetate, 1 ,7- dioxaspiro-(5-5) undecane, cis-7-tetradecenal, trans-8-dodecenyl acetate, cis-11-

hexadecenol, cis-13-octadecenal, trans-8-,trans-10-dodecadienol, cis-11 -hexadecenyl

acetate, 4-methyl-5-nonanol, [S]-verbenone, 3-methyl-2-cyclohexen-1-ol, 1-methyl-

butyl-2-trans-methyl-2-pentenoate, and 14-methyl-8-hexadecenal.

21. The pharmaceutical composition of claim 16 wherein the pheromone

compound is selected from the group consisting of cis-7-tetradecenyl, cis-11

tetradecenyl, trans-10-dodecenyl acetate, pentyl nonanoate,3,5-decadienyl acetate, 11 ,

11-difluoro-9-dodecenyl acetate, 9-tetradecenyl nitrate, 13-hexadecen-11-ynal, 9,12-

octadecadienal, 3,9-6,7-epoxydocosadiene, heptacosane, toluquinone, dodecalactone,

quinazolinone, cyclopentyl ketones, alkylpyrazines, indolizine, 9-oxo-2-decenoic acid,

pyrrolizidine, and castoramine.

22. A pharmaceutical composition comprising an effective amount of a

selected compound obtained by the method of claim 2 in combination with a

pharmaceutically acceptable carrier.

23. The pharmaceutical composition of claim 22 wherein the effective amount

is from about 0.1 to 200 mg/kg/day.

24. The pharmaceutical composition of claim 23 wherein the effective amount

is from about 2.0 to 180 mg/kg/day.

25. The pharmaceutical composition of claim 22 wherein the pheromone

7,cis-11-hexadecadienyl acetate, cis-11 -hexadecenal, cis-5-decenyl acetate, 4-methyl-

10-dodecadienol, cis-11 -hexadecenyl acetate, cis-11 -tetradecenal, 4-methyl-5-nonanol,

[S]-verbenone, 3-methyl-2-cyclohexen-1 -ol, 1 -methyl-butyl-2-trans-methyl-2-

acetate, cis-7-tetradecenyl acetate, and cis-9-heneicosene.

26. The pharmaceutical composition of claim 22 wherein the pheromone

compound is selected from the group consisting of trans-3-tridecenyl acetate, 1 ,7-

dioxaspiro-(5-5) undecane, cis-7-tetradecenal, trans-8-dodecenyl acetate, cis-11-

hexadecenol, cis-13-octadecenal, trans-8-,trans-10-dodecadienol, cis-11-hexadecenyl

butyl-2-trans-methyl-2-pentenoate, and 14-methyl-8-hexadecenal.

27. The pharmaceutical composition of claim 22 wherein the pheromone

compound is selected from the group consisting of cis-7-tetradecenyl, cis-11

pyrrolizidine, and castoramine.

28. A method of claim 1 wherein the desired therapeutic activity is selected

from the group consisting of prevention or treatment of cardiac arrhythmia, congestive

heart failure, vascular disease, hemorheological disease, multi-drug resistance,

impotence, female contraception, diabetes, neurological disorders, anxiety, and

erythropoietin stimulation.

29. A method of claim 2 wherein the desired therapeutic activity is selected

impotence, female contraception, diabetes, neurological disorders, anxiety, and

erythropoietin stimulation.

30. A method of providing a therapeutic activity in a warm-blooded animal

comprising administering to said warm-blooded animal an effective amount of the pharmaceutical composition of claim 16.

31. A method of providing therapeutic activity in a warm-blooded animal

comprising administering to said warm-blooded animal an effective amount of the

pharmaceutical composition of claim 22.

32. A pharmaceutical composition comprising an effective amount of at least

one pheromone compound selected from the group consisting of 1 ,2-hexadecanediol,

trans-5-decenyl acetate, trans-7,cis-9-dodecadienyl acetate, cis-7-tetradecenal, trans-5-

decenol, trans-8-,trans-10-dodecadienyl acetate, trans-8-dodecenyl acetate, cis-11-

dexadecenol, cis-7,cis-11-hexadecadienyl acetate, cis-11-hexadecenal, cis-5-decenyl

acetate, 4-methyl-pyrol-2-carboxylic acid methyl ester, trans-10, trans-12-

tetradecadienyl acetate, trans-11 -hexadecen-1 -yl acetate, trans-11 -hexadecen-1 -ol, cis-

3-dodecenyl-trans-2-butenoate, cis-7-tetradecenyl, cis-11 -tetradecenyl, cis-13-

octadecenal, trans-8-,trans-10-dodecadienol, cis-11-hexadecenyl acetate, cis-11-

tetradecenal,4-methyl-5-nonanol, [S]-verbenone, 3-methyl-2-cyclohexen-1-ol, 1-methyl-

butyl-2-trans-methyl-2-pentenoate, 14-methyl-8-hexadecenal, trans-,cis-3,7-

tetradecadienyl acetate, cis-7-tetradecenol, cis-7-dodecenyl acetate, cis-6-heneicosen-

11 -one, cis-4-tridecenyl acetate, cis-7-tetradecenyl acetate, and cis-9-heneicosene.

33. A method of treating a disease, condition or symptom thereof selected

from the group consisting of cardiac arrhythmia, congestive heart failure, vascular disease, hemorheological disease, multi-drug resistance, impotence, female

contraception, diabetes, neurological disorders, anxiety, and erythropoietin stimulation

comprising administering to said warm blooded animal an effective amount of at least

acetate, 4-methyl-pyrol-2-carboxylic acid methyl ester, trans-10, trans-12-

butyl-2-trans-methyl-2-pentenoate, 14-methyl-8-hexadecenal, trans-,cis-3,7-

11-one, cis-4-tridecenyl acetate, cis-7-tetradecenyl acetate, and cis-9-heneicosene.

34. A pharmaceutical composition comprising an effective amount of a

pheromone compound selected from the group consisting of cis-7-tetradecenyl, cis-11

quinazolinone, cyclopentyl ketones, alkylpyrazines, indolizine, 9-oxo-2-decenoic acid, pyrrolizidine, and castoramine.

35. A method of inducing MDR inhibiting activity in MDR cancer cells or MDR

microorganisms in a warm blooded animal comprising administering to said warm

blooded animal a pheromone compound selected from the group consisting of cis-7-

tetradecenyl, cis-11 tetradecenyl, trans-10-dodecenyl acetate, pentyl nonanoate,3,5-

decadienyl acetate, 11 , 11-difluoro-9-dodecenyl acetate, 9-tetradecenyl nitrate, 13-

hexadecen-11-ynal, 9,12-octadecadienal, 3,9-6,7-epoxydocosadiene, heptacosane,

toluquinone, dodecalactone, quinazolinone, cyclopentyl ketones, alkylpyrazines,

indolizine, 9-oxo-2-decenoic acid, pyrrolizidine, and castoramine.

36. A pharmaceutical composition comprising an effective amount of the

pheromone compound selected from the group consisting of trans-3-tridecenyl acetate,

1 ,7-dioxaspiro-(5-5) undecane, cis-7-tetradecenal, trans-8-dodecenyl acetate, cis-11 -

butyl-2-trans-methyl-2-pentenoate, and 14-methyl-8-hexadecenal.

37. A method of modulating cGMP in a warm-blooded animal comprising

administering to said warm-blooded animal an effective amount of the pharmaceutical

composition of claim 36.

38. A pharmaceutical composition comprising an effective amount of the

pheromone compound selected from the group consisting of 1 ,2-hexadecanediol, 1 ,2-

octadecanediol, and 15-methyl-1 ,2-hexadecanediol.

39. A method of preventing or treating cardiac arrhythmia in a warm-blooded

animal comprising administering to said warm-blooded animal an effective amount of

the pharmaceutical composition of claim 38.