WO2006039285A2 - Compositions and methods for inducing adipose tissue cell death - Google Patents

Abstract

Methods of treating obesity, methods for inducing apoptosis in adipose tissue cells in a host, pharmaceutical compositions, and methods for treating osteoporosis in a host, are disclosed.

Description

COMPOSITIONS AND METHODS FOR INDUCING ADIPOSE TISSUE CELL DEATH

CROSS REFERFENCE TO RELATED APPLICATION

The present application claims priority to and the benefit of U.S. provisional

patent application No. Serial 60/614,738, filed on September 30, 2004 and entitled

"Compositions and Methods for Inducing Adipose Tissue Cell Death", which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present disclosure is generally related to compositions and methods for

administration of the compositions to hosts and, more particularly, is related to

compositions designed for treatment for inducing adipose tissue cell death and

methods of administration thereof.

BACKGROUND

Obesity represents a major public health issue that continues to grow, accounts

for 5.7% of total direct health care costs in the United States, and increases the risk of acquiring a disease that is a leading cause of death {e.g., cardiovascular disease,

diabetes, and cancer). Obesity is marked by excess adipose (i.e., fat) tissue

accumulation, which arises from both an increase in number and size of adipocytes

due to higher levels of lipid storage. Excess adipose tissue is strongly correlated with

numerous health problems, including diabetes (e.g., decreased insulin sensitivity),

vascular disease (e.g., hypertension), and certain forms of cancer. Exacerbating the health risks associated with obesity, the most popular treatment for morbid obesity, liposuction, is a largely unregulated, half-billion dollar

industry that exposes patients to additional health risks, including infection and death.

As the current strategy for adipose reduction, liposuction is an invasive and painful

procedure requiring costly equipment and considerable recovery time that often results

in inconsistent tissue shape. Gastric bypass surgery is another extreme treatment for

obesity that is rapidly gaining popularity. Like liposuction, gastric bypass surgery is fraught with risks. It is an even more invasive and serious procedure that requires

extensive recovery time, has a high risk of complications, including death, both during and after surgery, and is too expensive for most potential candidates. Thus, a less

invasive, safer, less expensive, and more reliable way of treating obesity is needed.

Approximately 10 million people in the U.S. are estimated to have

osteoporosis, a disease that results in over 1.5 million bone fractures a year. The

direct expenditures for osteoporosis in 2001 totaled $17 billion, which equals a cost of

$47 million per day. Osteoporosis is therefore a significant health problem, and

effective ways to treat and prevent osteoporosis are needed.

SUMMARY

Methods of treating obesity, methods for inducing apoptosis in adipose tissue

cells in a host, pharmaceutical compositions, and methods for treating osteoporosis in

a host, are disclosed. An illustrative embodiment of a method of treating obesity,

among others, includes administering to a host in need of treatment an effective

amount of a compound selected from: ajoene, precursors thereof, and derivatives

thereof. The compound is present in a dosage level effective to initiate the release or

activation of an apoptosis inducing factor. The apoptosis inducing factor leads to the

apoptotic cell death of adipose tissue cells in the host. An illustrative embodiment of a method for inducing apoptosis in adipose

tissue cells in a host, among others, includes initiating the release or activation of an

apoptosis inducing factor by administering an effective amount of at least one garlic

extract compound. The apoptosis inducing factor leads to apoptotic cell death.

An illustrative embodiment of a pharmaceutical composition, among others,

includes at least one garlic extract compound in combination with a pharmaceutically

acceptable carrier. The at least one garlic extract compound is present in a dosage

level effective to initiate the release of an apoptosis inducing factor. The apoptosis inducing factor leads to the apoptotic cell death of adipose tissue cells in a host.

An illustrative embodiment of a method for treating osteoporosis in a host,

among others, includes administering an effective amount of at least one garlic extract

compound to a host in need of treatment.

An illustrative embodiment of a method for treating osteoporosis in a host,

among others, includes administering to a host an effective amount of a

pharmaceutical composition including at least one garlic extract compound in combination with a pharmaceutically acceptable carrier. The garlic extract compound

is present in a dosage level effective to treat osteoporosis.

Other compositions, methods, features, and advantages of the present

disclosure will be or become apparent to one with skilled in the art upon examination

of the following drawings and detailed description. It is intended that all such

additional compositions, methods, features, and advantages be included within this

description, be within the scope of the present disclosure, and be protected by the

accompanying claims. BRIEF DESCRIPTION OF THE DRAWINGS

Many aspects of the disclosure can be better understood with reference to the

following drawings.

FIG. 1 illustrates the effect of ajoene on cell viability. 3T3-L1 adipocytes were

incubated with ajoene at various concentrations (0, 50, 100, 200, 400 μM) for 6, 12

and 24 h. Cell viability after ajoene treatment was determined by the MTS

colorimetric assay. Assays were performed in eight replicates for each treatment. Within a time period, means with different letters are different, abc: p<0.05; wxyz:

p<0.01.

FIGS. 2A and 2B illustrate the effect of ajoene on apoptosis. In FIG. 2A 3T3-

Ll adipocytes were treated with ajoene at various concentrations (0, 10, 50, 200, 400

μM) for 3, 6, 12 and 24 h. Cells were fixed and analyzed by ssDNA ELISA. Assays

were performed in eight replicates for each treatment. Within a time period, means

with different letters are different, xy: p<0.01. FIG. 2B illustrates apoptosis by gel

electrophoresis. 3T3-L1 adipocytes were incubated with ajoene at various

concentrations (0, 100, 200, 400 μM) for 24 hr. Total cellular DNA was extracted and

analyzed by gel electrophoresis; Lane 1, control; Lane 2, 100 μM ajoene; Lane 3, 200

μM ajoene; Lane 4, 400 μM ajoene; Lane 5, DNA Marker.

FIG. 3 A and 3B illustrate the effects of ajoene on intracellular hydrogen

peroxide production. FIG. 3 A illustrates the time course of the effect of ajoene on

ROS production. 3T3-L1 adipocytes were grown in 96-well plates and treated with

200 μM ajoene, and the change in fluorescence of the oxidized probe was determined.

Each value is the mean ± SEM of eight replicates. Means with different letters are

different (vwxyz), p<0.01. FIG. 3B illustrates the reduction of ajoene-induced apoptosis in cells pretreated with NAC. The percentage of apoptotic cells measured

as described in FIG. 2A is shown for untreated cells (control); cells exposed to ajoene

(200 μM, 24hr); cells preloaded with 10 mM NAC for Ih, either exposed or not

exposed to ajoene (200 μM, 24hr); and cells preloaded with catalase (400 units/ml) in

the absence or presence of ajoene (200 μM, 24hr). Assays were performed in eight

replicates for each treatment. Means with different letters are different (x,y), p<0.01.

FIGS. 4A-4D illustrate the effect of ajoene on MAPKs phosphorylation. The protein levels of unphosphorylated and phosphorylated forms of MAP kinases were

evaluated in cytosolic proteins by Western blotting with the use of specific antibodies.

For the experiments shown in FIG. 4A and 4B, 3T3-L1 adipocytes were treated with

ajoene (200 μM) for the indicated time periods. Representative Western blots show

the time-dependent phosphorylation of (FIG. 4A) JNK and (FIG. 4B) ERK1/2 in the

upper panels and the expression levels of the respective total kinases in the lower

panels.

For the experiments shown in FIG. 4C and FIG. 4D, 3T3-L1 adipocytes were

preincubated with 10 mM NAC for 1 hr before ajoene (200 μM) treatment for either

30 min (for ERK1/2) or 3 hours (for JNK). Western blots showed ERK 1/2 and JNK

phosphorylation (upper panels) as well as the respective total kinases (lower panels).

Densitometric quantitation of the autoradiograms for phosphorylated and total

ERKl/2 and JNK were performed. Integrated density values (phosphorylated/total)

were calculated and expressed as % 0 h (A & B) or % of control (C & D). AU

experiments were repeated two to four times. The data were analyzed by ANOVA

followed by post-hoc analysis by LSD tests. Means with different letters are different, abc: p<0.05; xyz: pθ.01. FIGS. 5A-5C illustrate the effect of ajoene on PARP cleavage and caspase3

activation. In FIG. 5A 3T3-L1 adipocytes were treated with ajoene (200 μM) for the

indicated time periods. Cell lysates were analyzed by Western blotting, and PARP

(upper panel) and caspase-3 protein band (middle panel) were detected using specific

antibodies. β-Actin (lower panel) was used as an equal loading control.

Densitometric quantitation of the autoradiograms for PARP and caspase-3 were

performed. Integrated density values (PARP or caspase/actin) were calculated and expressed as % highest value. All experiments were repeated two to-four times. The

data were analyzed by ANOVA followed by posthoc analysis by LSD tests. Means

with different letters are different: abc, p<0.05; xyz, p<0.01.

In FIG. 5B 3T3-L1 adipocytes were preincubated with 10 mM NAC for 1 hr

before ajoene treatment (200 μM, 12hr). Cell lysates were analyzed by Western

blotting and PARP protein band (upper panel) was detected using specific antibodies.

β-Actin (lower panel) was used as an equal loading control. Densitometric

quantitation of the autoradiogram of PARP was performed. Integrated density values

(PARP/actin) were calculated and expressed as % highest value. AU experiments

were repeated two to four times. The data were analyzed by ANOVA followed by posthoc analysis by LSD tests. Means with different letters are different, xy: p<0.01.

In FIG. 5C 3T3-L1 adipocytes were treated with ajoene at various

concentrations (0, 50, 100, 200 μM) for 1, 3, 6, 12 and 24 hr. Cells were assayed for

caspase-3 activity. Assays were performed in eight replicates for each treatment.

Within a time period, means with different letters are different, xyz: p<0.01.

FIGS. 6A-6B illustrate ajoene induced translocation of AIF from mitochondria

to nucleus. In FIG. 6A 3T3-L1 adipocytes were treated with ajoene (200 μM) for 0, 1, 4, 8 and 12 hr. Equal amounts of AIF protein from nuclear fraction (upper panel) and

mitochondrial fraction (lower panel) were analyzed by Western blotting using an anti-

AIF antibody. Densitometric quantitation of the autoradiogram was performed.

Integrated density values (AIF/actin) were calculated and expressed as % highest

value. Experiments were repeated two times. The data were analyzed by ANOVA

followed by posthoc analysis by LSD tests. Within an AIF source (nuclear or

mitochondrial) means with different letters are different across time: xyz: p<0.01.

In FIG. 6B 3T3-L1 adipocytes were preincubated with 10 mM NAC for 1 hr in

the absence or presence of ajoene (200 μM, 8 hr). Cell lysates were analyzed by

Western blotting using the ami- AIF antibody (upper panel) and β-Actin (lower panel),

which served as an internal control. Densitometric quantitation of the autoradiogram

was performed. Integrated density values (AIF/actin) were calculated and expressed

as % control. Experiments were repeated two times. The data were analyzed by

ANOVA followed by posthoc analysis by LSD tests. Means with different letters are different: xy: p<0.01.

DETAILED DESCRIPTION

Embodiments of the present disclosure will employ, unless otherwise indicated,

techniques of synthetic organic chemistry, biochemistry, molecular biology, and the like,

that are within the skill of the art. Such techniques are explained fully in the literature.

The following examples are put forth so as to provide those of ordinary skill in

the art with a complete disclosure and description of how to perform the methods and

use the compositions disclosed and claimed herein. Efforts have been made to ensure

accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors

and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard

temperature and pressure are defined as 20 °C and 1 atmosphere.

Before the embodiments of the present disclosure are described in detail, it is

to be understood that, unless otherwise indicated, the present disclosure is not limited

to particular materials, reagents, reaction materials, manufacturing processes, or the

like, as such can vary. It is also to be understood that the terminology used herein is

for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in

different sequence where this is logically possible.

It must be noted that, as used in the specification and the appended claims, the

singular forms "a," "an" and "the" include plural referents unless the context clearly

dictates otherwise. Thus, for example, reference to "a support" includes a plurality of

supports.

Definitions:

hi this specification and in the claims that follow, reference will be made to a

number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

The term "organism" or "host" refers to any living entity comprised of at least

one cell. A living organism can be as simple as, for example, a single eukaryotic cell

or as complex as a mammal, including a human being. As used herein, the term

"host" includes humans, mammals (e.g., cats, dogs, horses, chicken, pigs, hogs, cows,

and other cattle), and other living species that are in need of treatment, hi particular,

the term "host" includes humans, companion animals (e.g., cats, dogs, and the like),

and livestock animals (e.g., pigs, cattle, and the like). The term "apoptosis inducing factor" as used herein refers to any compound

activated in a pathway leading to apoptosis. For instance, "apoptosis inducing factor"

includes not only the Apoptosis Inducing Factor protein (AIF), but also any compound

upstream in the pathway leading to ADF release and/or activation, as well as other

compounds which, when activated in certain amounts or under certain conditions lead

to apoptosis. Exemplary apoptosis inducing factors in the present disclosure include,

but are not limited to, reactive oxygen species (ROS), mitogen-activated protein kinases (MAPKs) such as ERK1/2 and INK, PoIy(ADP ribose) polymerases (PARPs),

and AIF. However, apoptosis inducing factors as used in the present disclosure do not

include caspases.

The term "derivative" refers to a modification to the disclosed compounds.

The term "therapeutically effective amount" as used herein refers to that

amount of the compound being administered that will relieve to some extent one or

more of the symptoms of the disorder being treated, hi reference to adipose tissue, a

therapeutically effective amount refers to that amount that has the effect of (1) causing

apoptosis of adipose cells and/or (2) reducing the mass of the adipose cells/tissue.

"Pharmaceutically acceptable salt" refers to those salts that retain the

biological effectiveness and properties of the corresponding free bases and that are

obtained by reaction with inorganic or organic acids such as hydrochloric acid,

hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid,

ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, malic acid, maleic acid,

succinic acid, tartaric acid, citric acid, and the like.

The term "pharmaceutically acceptable esters" as used herein refers to those

esters of one or more compounds of the composition that, within the scope of sound

medical judgment, are suitable for use in contact with the tissues of hosts without undue toxicity, irritation, allergic response, and the like, are commensurate with a

reasonable benefit/risk ratio, and are effective for their intended use.

A "pharmaceutical composition" refers to a mixture of one or more of the

compounds described herein, derivatives thereof, or pharmaceutically acceptable salts

thereof, with other chemical components, such as pharmaceutically acceptable carriers

and excipients. One purpose of a pharmaceutical composition is to facilitate

administration of a compound to the organism.

As used herein, a "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate

the biological activity and properties of the administered compound.

An "excipient" refers to an inert substance added to a pharmaceutical

composition to further facilitate administration of a compound. Examples of

excipients include, without limitation, calcium carbonate, calcium phosphate, various

sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and

polyethylene glycols.

"Treating" or "treatment" of a condition includes preventing the condition

from occurring in an animal that may be predisposed to the disease but does not yet experience or exhibit symptoms of the condition (prophylactic treatment), inhibiting

the condition (slowing or arresting its development), providing relief from the

symptoms or side-effects of the condition (including palliative treatment), and

relieving the condition (causing regression of the condition).

The term "prodrug" refers to an agent that is converted into a biologically

active form in vivo. Prodrugs are often useful because, in some situations, they may

be easier to administer than the parent compound. They may, for instance, be

bioavailable by oral administration whereas the parent compound is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent

drug. A prodrug may be converted into the parent drug by various mechanisms,

including enzymatic processes and metabolic hydrolysis. Harper, NJ. (1962). Drug

Latentiation in Jucker, ed. Progress in Drug Research, 4:221-294; Morozowich et al.

(1977). Application of Physical Organic Principles to Prodrug Design in E. B. Roche

ed. Design of Biopharmaceutical Properties through Prodrugs and Analogs, APhA;

Acad. Pharm. ScL; E. B. Roche, ed. (1977). Bioreversible Carriers in Drug in Drug Design, Theory and Application, APhA; H. Bundgaard, ed. (1985) Design of Prodrugs, Elsevier; Wang et al. (1999) Prodrug approaches to the improved delivery

of peptide drug, Curr. Pharm. Design. 5(4):265-287; Pauletti et al. (1997).

Improvement in peptide bioavailability: Peptidomimetics and Prodrug Strategies, Adv.

Drug. Delivery Rev. 27:235-256; Mizen et al. (1998). The Use of Esters as Prodrugs

for Oral Delivery of β-Lactam antibiotics, Pharm. Biotech. ll,:345-365; Gaignault et

al. (1996). Designing Prodrugs and Bioprecursors I. Carrier Prodrugs, Pract. Med.

Chem. 671-696; M. Asgharnejad (2000). Improving Oral Drug Transport Via

Prodrugs, in G. L. Amidon, P. I. Lee and E. M. Topp, Eds., Transport Processes in Pharmaceutical Systems, Marcell Dekker, p. 185-218; Balant et al. (1990) Prodrugs

for the improvement of drug absorption via different routes of administration, Eur. J.

DrugMetab. Pharmacokinet., 15(2): 143-53; Balimane and Sinko (1999).

Involvement of multiple transporters in the oral absorption of nucleoside analogues,

Adv. Drug Delivery Rev., 39(l-3):183-209; Browne (1997). Fosphenytoin (Cerebyx),

CHn. Neuropharmacol. 20(1): 1-12; Bundgaard (1979). Bioreversible derivatization

of drugs— principle and applicability to improve the therapeutic effects of drugs, Arch.

Pharm. Chemi. 86(1): 1-39; H. Bundgaard, ed. (1985), Design of Prodrugs, New

York: Elsevier; Fleisher et al. (1996). Improved oral drug delivery: solubility limitations overcome by the use of prodrugs, Adv. Drug Delivery Rev. 19(2): 115-130; Fleisher et al. (1985). Design of prodrugs for improved gastrointestinal absorption by

intestinal enzyme targeting, Methods Enzymol. 112: 360-81; Farquhar D, et al. (1983).

Biologically Reversible Phosphate-Protective Groups, J. Pharm. ScL, 72(3): 324-325;

Han, H.K. et al. (2000). Targeted prodrug design to optimize drug delivery, AAPS

PharmScL, 2(1): E6; Sadzuka Y. (2000). Effective prodrug liposome and conversion to active metabolite, Curr. DrugMetab., l(l):31-48; D.M. Lambert (2000). Rationale

and applications of lipids as prodrug carriers, Eur. J. Pharm. ScL, 11 Suppl 2:S15-27; Wang, W. et al. (1999) Prodrug approaches to the improved delivery of peptide drugs.

Curr. Pharm. Des., 5(4):265-87.

As used herein, the term "topically active agents" refers to compositions of the

present disclosure that elicit pharmacological responses at the site of application

(contact) to a host.

As used herein, the term "topically" refers to application of the compositions

of the present disclosure to the surface of the skin and mucosal cells and tissues.

The disclosed compounds can form salts that are also within the scope of this

disclosure. Reference to each compound herein is understood to include reference to

salts thereof, unless otherwise indicated. The term "salt(s)," as employed herein,

denotes acidic and/or basic salts formed with inorganic and/or organic acids and

bases. In addition, when a compound contains both a basic moiety and an acidic

moiety, zwitterions ("inner salts") may be formed and are included within the term

"salt(s)" as used herein. Pharmaceutically acceptable {i.e., non-toxic, physiologically

acceptable) salts are preferred, although other salts are also useful {e.g., in isolation or

purification steps which may be employed during preparation). Salts of the

compounds may be formed, for example, by reacting the compound with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt

precipitates or in an aqueous medium followed by lyophilization.

The disclosed compounds that contain a basic moiety may form salts with a

variety of organic and inorganic acids. Exemplary acid addition salts include acetates

(such as those formed with acetic acid or trihaloacetic acid, for example,

trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates,

benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates,

heptanoates, hexanoates, hydrochlorides (formed with hydrochloric acid),

hydrobromides (formed with hydrogen bromide), hydroiodides, 2-

hydroxyethanesulfonates, lactates, maleates (formed with maleic acid),

methanesulfonates (formed with methanesulfonic acid), 2-naphthalenesulfonates,

nicotinates, nitrates, oxalates, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates (such as

those formed with sulfuric acid), sulfonates (such as those mentioned herein),

tartrates, thiocyanates, toluenesulfonates (such as tosylates), undecanoates, and the

like.

The disclosed compounds that contain an acidic moiety may form salts with a

variety of organic and inorganic bases. Exemplary basic salts include ammonium

salts; alkali metal salts such as sodium, lithium, and potassium salts; alkaline earth

metal salts such as calcium and magnesium salts; salts with organic bases (for

example, organic amines) such as benzathines, dicyclohexylamines, hydrabamines

(formed with N,N-bis(dihydroabietyl)ethylenediamine), N-methyl-D-glucarnines, N- methyl-D-glucamides, t-butyl amines; and salts with amino acids such as arginine,

lysine, and the like.

Basic nitrogen-containing groups may be quaternized with agents such as

lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides and

iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and diamyl sulfates), long

chain halides (e.g., decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides),

aralkyl halides (e.g., benzyl and phenethyl bromides), and others.

Solvates of the compounds are also contemplated herein. Solvates of the compounds are preferably hydrates.

To the extent that the disclosed compounds, and salts thereof, may exist in

their tautomeric form, all such tautomeric forms are contemplated herein as part of the

present disclosure.

All stereoisomers of the present compounds, such as those which may exist

due to asymmetric carbons on the various substituents, including enantiomeric forms

(which may exist even in the absence of asymmetric carbons) and diastereomeric

forms, are contemplated within the scope of this disclosure. Individual stereoisomers of the compounds of the disclosure may, for example, be substantially free of other

isomers, or may be admixed, for example, as racemates or with all other, or other

selected, stereoisomers. The chiral centers of the compounds of the present disclosure

can have the S or R configuration as defined by the IUPAC 1974 Recommendations.

Discussion

Embodiments of the present disclosure provide for pharmaceutical

compositions, methods for inducing adipose tissue cell death, and methods for treating

obesity. In particular, the compositions include, but are not limited to, compositions having at least one garlic extract compound and, in particular, garlic thiosulfinates and

transformation products thereof. In addition, the method includes inducing adipose

tissue cell death in a host by administering a composition having at least one garlic

extract compound. Further, the methods include treating conditions such as, but not

limited to, obesity and/or osteoporosis, in a host with compositions having at least one

garlic extract compound.

Obesity is a chronic and costly condition that is increasing rapidly throughout the world. Obesity is considered a major risk factor for noninsulin-dependent diabetes

mellitus (1) and has also been linked to cancer and immune dysfunction (2). Adipose tissue mass is determined by processes governing adipocyte size and number (3).

Reduction of adipocyte number can result from preadipocyte and adipocyte apoptosis,

as well as adipocyte de-differentiation (4). Therefore, apoptosis may be an important

mechanism regulating adipose tissue mass.

The garlic extract compound of the present disclosure (e.g., ajoene)

unexpectantly and surprisingly initiates the release of an apoptosis inducing factor,

where the apoptosis inducing factor leads to the apoptotic cell death of adipose tissue cells. This apoptotic cell death is unexpectedly caspase-independent, which is in

contrast to the general understanding that caspases are the primary mediators of

apoptosis under most circumstances. The caspase-independent apoptotic pathway

induced by the compounds of the present disclosure is discussed in more detail in the

Example.

In general, apoptosis refers to a physiological process wherein selected cells

are deleted in a rapid, efficient fashion through a signal-induced activation of

endogenous self-destructive cellular processes. Apoptosis involves a sequence of

distinct biochemical and morphological events characterized by DNA fragmentation, cell volume shrinkage, and production of plasma membrane-bounded apoptosis

bodies, ultimately leading to cell death.

Several organosulfur compounds from garlic, including allicin and its

derivatives, have inhibited the proliferation of some tumor cell lines (5-7). Ajoene is

a garlic-derived compound and has a greater chemical stability than allicin. Ajoene

was shown to induce apoptosis in human promyeloleukemic cells via the activation of

nuclear factor KB (8).

ROS are recognized to play a key role in cell signaling. At the cellular level,

oxidant injury elicits a broad spectrum of responses ranging from proliferation to growth arrest, to senescence, to cell death (for review see 9). Activation of mitogen-

activated protein kinases (MAPK) is considered to be a pivotal step in ROS-induced

signaling pathways. It has been shown that increased ROS production in leukemic

cells leads to the activation of MAPK and cell death (10-13). The MAPK pathways

include three parallel kinase modules, namely, the extracellular signal-regulating

kinase (ERKl/2), the Jun-N-teraiinal kinase (JNK), and the p38 MAPK pathways. In

general, JNK and p38 MAPK activation is associated with apoptosis induction, whereas ERK activation is cytoprotective (14).

Single-strand breaks in DNA, resulting from free radical and oxidant cell

injury, can trigger the activation of the nuclear enzyme, PoIy(ADP ribose) polymerase

(PARP), which contributes to the pathogenesis of various diseases (15, 16). PARP is

a highly conserved, 113 kDa nuclear enzyme which, when activated, generates a

cleaved 85 kDa PARP product.

Apoptosis Inducing Factor (AIF) was more recently cloned and identified as a

mitochondrial intermembrane space protein. In response to apoptotic stimuli, ATF is

released from mitochondria and translocated to the nucleus where it participates in the induction of chromatin condensation, the exposure of phosphatidyl-serine in the outer

leaf of the plasma membrane, and the dissipation of the mitochondrial transmembrane

potential, ultimately resulting in apoptosis (17). Recently, the importance of AIF in

oxidant-induced cell injury in neurons was demonstrated (18). The apoptotic pathway

in the response of 3T3-L1 adipocytes to agents such as ajoene has not previously been

explored, nor have the relationships between components of this pathway.

hi particular, the induction of apoptosis of adipose tissue cells according to the

methods and compounds of the present disclosure is unexpected in that it occurs via the release of apoptosis inducing factors as opposed to the release of caspases. As discussed in more detail in the Example, ajoene-induced apoptosis in 3T3-L1

adipocytes is initiated by the generation of hydrogen peroxide, which leads to

activation of MAPKs, degradation of PARP-I, translocation of AIF, fragmentation of

DNA, and adipose tissue cell death. Therefore, ajoene, a garlic extract compound, can

influence the regulation of fat cell number in a host through the induction of apoptosis

in adipose tissue cells.

As described in more detail in the Example, mature 3T3-L1 adipocytes were

incubated with ajoene at concentrations up to 200 μM. Viability and apoptosis were

quantified using MTS and an ELISA assay for single stranded DNA (ssDNA),

respectively. Intracellular reactive oxygen species (ROS) production was measured

based on production of the fluorescent dye, DCF. Activation of the mitogen-activated

protein kinases (MAPK), extracellular signal-regulating kinase 1/2 (ERK), and c-Jun-

N-terminal kinase (JNK) was demonstrated by Western blot. Western blot was also used to demonstrate activation of caspase-3, translocation of apoptosis inducing factor

(ATF) from mitochondria to nucleus, and cleavage of 116 kDa poly(ADP-ribose)

polymerase (PARP)-I. Ajoene induced apoptosis of 3T3-L1 adipocytes in a dose-and time-dependent

manner. Ajoene treatment resulted in activation of JNK and ERK, translocation of

AIF from mitochondria to nucleus, and cleavage of 116 kDa PARP-I in a caspase-

independent manner. Ajoene treatment also induced an increase in intracellular

reactive oxygen species (ROS) level but did not result in activation of caspases.

Further, the antioxidant N-acetyl-_L-cysteine (NAC) effectively blocked ajoene-

mediated'ROS generation, activation of JNK and ERK, translocation of AIF, and degradation of PARP-I. Additional details are described in the Example.

To detect apoptosis, several techniques have been developed based on the understanding of the morphological, biochemical and molecular mechanisms

involved. Changes in the phospholipid bilayers of cell membranes are observed early

in the apoptosis process. The phosphatidylserine (PS) component of the phospholipid

bilayer is externalized and can be detected by fluorescence labeling. Annexin V (AV),

a member of the annexin family of calcium-dependent phospholipid-binding proteins,

has a high affinity for PS-containing phospholipid bilayers. FITC-conjugated AV is

used as a fluorescent dye to detect this early event in apoptotic cells.

As the apoptotic process progresses, cell membranes lose integrity, allowing

chromosomal DNA to be exposed. Propidium iodide, a fluorescent dye that binds to

DNA can be used in conjunction with FITC-conjugated AV to identify subpopulations

of cells with end-stage apoptotic changes.

The TUNEL enzymatic labeling assay is another method used to detect

apoptosis in individual cells. Extensive DNA fragmentation/degradation is a

characteristic event that occurs in apoptosis. The TUNEL assay is used to detect

DNA strand breaks by labeling the free 3'-OH ends. Both of these assays can be used in conjunction with laser scanning cytometry (LSC) to provide both quantitative and morphological analysis of apoptosis. Laser

Scanning Cytometry uses lasers to excite fluorochromes in cellular specimens and

detects the fluorescence in discrete wavelengths with multiple photomultiplier tubes

(PMT's). Data are collected on heterogeneous populations of cells, and software

analysis tools are used to obtain statistical analysis of the populations. LSC also

creates temporary digital images of the specimens on microscope slides and employs image processing algorithms to identify and segment the "events" (e.g., individual

cells). LSC can additionally find and quantitate events by multiple filter settings, for example, making it possible to distinguish cytoplasmic fluorescence from nuclear

fluorescence. Finally, LSC generates high-resolution images that allow visual

inspection of individual cells of interest.

LSC has been used to study apoptosis of adipocytes and has been shown to

provide a relatively fast method for obtaining both quantitative and morphological

information about the apoptotic process in adipocytes.

The two dyes used, Annexin V (AV) and propidium iodide (PI), are

fluorescent dyes that have different absorption spectra, so by using 2 lasers with

different wave lengths, both dyes can be detected at the same time in a single sample. The microscope stage moves the slide (e.g., incubation dish) automatically through

the laser beams. For each segment that is scanned, the computer stores information

about the intensity of the fluorescence from each dye. It also stores digital images of

each segment, which allows one to go back and pick out a point on the scattergraph

and look to see the cells that generated a specific data point.

Another method to detect apoptosis is based on the staining of cell suspensions

and tissue sections with monoclonal antibodies (MAbs) to single-stranded DNA (ssDNA) (Methods MoI Biol 282: 85-102 (2004)). The higher sensitivity of MAb

staining compared to TUNEL reflects the different mechanisms of the two techniques.

TUNEL detects low-mol-wt DNA fragmentation associated with late apoptosis,

whereas MAbs to ssDNA detect the early stages of apoptosis and stain apoptotic cells

in the absence of low-mol-wt DNA fragmentation (Methods MoI Biol 282: 85-102

(2004); Anticancer Res 14(5A): 1861-9 (1994); Exp Cell Res 226(2): 387-97 (1996)). These advantages of the MAb method are based on the fact that protease activation is

an early and universal event in apoptosis (Cell 82(3): 349-52 (1995)). Importantly, in contrast with the TUNEL method, MAbs to ssDNA are specific for apoptotic cell

death and do not detect necrotic cells (Methods MoI Biol 282: 85-102 (2004);

Anticancer Res 14(5A): 1861-9 (1994); Exp Cell Res 226(2): 387-97 (1996)).

In regard to osteoporosis, it is now known that the accumulation of fat cells

(adipocytes) in bone marrow is a major factor contributing to age-related bone loss.

Women with osteoporosis have higher numbers of marrow adipocytes than women

with healthy bone (Clin Orthop 80: 147-54 (1971); J Bone Miner Res 12(11): 1772-9 (1997); and Bio gerontology 2(3): 165-71 (2001)), and bone formation rate is inversely

correlated with adipocyte number in bone tissue biopsies from both men and women

(J Clin Pathol 55(9): 693-8 (2002)). Recent in vivo and in vitro studies provide

important insights into why marrow adipogenesis is associated with bone loss. First,

mesenchymal stem cells within bone marrow can differentiate to form adipocytes or

osteoblasts. Conditions favoring adipocyte differentiation will therefore have adverse

effects on bone formation because precursor cells are directed towards the adipocyte

lineage rather than the osteoblast lineage (J Musculoskel Neuron Interact 2: 581-583

(2002); J Clin Invest 113(6): 846-55 (2004)). Second, adipocytes secrete

osteoclastogenic cytokines such as JX-6 (J Clin Endo Metab 83(3): 847-850 (1998), and adipocytes can inhibit osteoblast activity in culture (^"Bone 26(5): 485-9 (2000)).

Finally, fat cell development and hypertrophy can compress intraosseous capillaries,

which decreases blood supply within bone (Joint Bone Spine 69(3): 262-9 (2002)).

Thus, removal of adipocytes from bone marrow through induction of apoptosis could

prevent or reverse bone loss associated with osteoporosis.

The present disclosure provides compounds, such as, garlic extract

compounds, for inducing apoptosis of adiposites. Garlic extract compounds of the

present disclosure can include, but are not limited to, garlic thiosulfinates and transformation products thereof, ajoene (E and Z isomers) (4,5,9-trithiadodeca-l,6,ll- triene-9-oxide), precursors thereof, and derivatives thereof; allicin, precursors thereof,

and derivatives thereof; allyl methanethiosulfinate, precursors thereof, and derivatives

thereof; disulfides, precursors thereof, and derivatives thereof; allylsulfides,

precursors thereof, and derivatives thereof; vinyldithiins, precursors thereof, and

derivatives thereof; diallyl trisulfides, precursors thereof, and derivatives thereof; and

mercaptocysteines, precursors thereof, and derivatives thereof; and combinations

thereof, hi addition, the garlic extract compound can include, but is not limited to, S-

allylmercaptocysteme (SAMC), S-methylcysteine (SMC), S-allyl cysteine sulfoxide (SACS), diallyl disulfide (DADS), S-allylcysteine (SAC), S-ethylcysteine (SEC), S-

propylcysteine (SPC), and combinations thereof, hi one embodiment, the garlic

extract compound includes ajoene (E and Z isomers), and/or precursors thereof, and/or

derivatives thereof.

Where such forms exist, garlic extract compounds may include garlic extract compound analogues, homologues, isomers, or derivatives thereof, that function to

induce adipose tissue cell death in a caspase-independent manner. In addition, garlic extract compounds can include pharmaceutically acceptable salts, esters, and prodrugs of the garlic extract compounds described above.

Embodiments of the present disclosure include methods for inducing apoptosis

of adipose cells and/or reducing the mass of adipose cells/tissue in a host, m addition,

embodiments of this disclosure include methods to treat conditions such as, but not

limited to, obesity, osteoporosis and related conditions and diseases in a host.

Pharmaceutical compositions and dosage forms of the disclosure include a

pharmaceutically acceptable salt of the compound and/or a pharmaceutically acceptable polymorph, solvate, hydrate, dehydrate, co-crystal, anhydrous, or

amorphous form thereof. Specific salts of disclosed compounds include, but are not

limited to, sodium, lithium, and potassium salts, and hydrates thereof.

Pharmaceutical unit dosage forms of the compounds of this disclosure are

suitable for oral, mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral

(e.g., intramuscular, subcutaneous, intravenous, intra-arterial, or bolus injection),

topical, or transdermal administration to a patient. Examples of dosage forms include,

but are not limited to: tablets; caplets; capsules, such as hard gelatin capsules and soft

elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories;

ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters;

solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms

suitable for oral or mucosal administration to a patient, including suspensions (e.g.,

aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil

liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral

administration to a patient; and sterile solids (e.g., crystalline or amorphous solids)

that can be reconstituted to provide liquid dosage forms suitable for parenteral

administration to a patient. The composition, shape, and type of dosage forms of the compositions of the

disclosure typically vary depending on their use. For example, a dosage form used in

the acute treatment of a condition or disorder may contain larger amounts of the active

ingredient, e.g., the disclosed compounds or combinations thereof, than a dosage form used in the chronic treatment of the same condition or disorder. Similarly, a

parenteral dosage form may contain smaller amounts of the active ingredient than an

oral dosage form used to treat the same condition or disorder. These and other ways

in which specific dosage forms encompassed by this disclosure vary from one another will be readily apparent to those skilled in the art (See, e.g., Remington's

Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton, Pa. (1990)).

Typical pharmaceutical compositions and dosage forms include one or more

excipients. Suitable excipients are well known to those skilled in the art of pharmacy

or pharmaceutics, and non-limiting examples of suitable excipients are provided

herein. Whether a particular excipient is suitable for incorporation into a

pharmaceutical composition or dosage form depends on a variety of factors well

known in the art including, but not limited to, the way in which the dosage form will

be administered to a patient. For example, oral dosage forms, such as tablets or

capsules, may contain excipients not suited for use in parenteral dosage forms. The

suitability of a particular excipient may also depend on the specific active ingredients

in the dosage form. For example, the decomposition of some active ingredients can

be accelerated by some excipients, such as lactose, or by exposure to water. Active

ingredients that include primary or secondary amines are particularly susceptible to

such accelerated decomposition.

The disclosure further encompasses pharmaceutical compositions and dosage

forms that include one or more compounds that reduce the rate by which an active ingredient will decompose. Such compounds, which are referred to herein as

"stabilizers," include, but are not limited to, antioxidants such as ascorbic acid, pH

buffers, or salt buffers. In addition, pharmaceutical compositions or dosage forms of

the disclosure may contain one or more solubility modulators, such as sodium

chloride, sodium sulfate, sodium or potassium phosphate, or organic acids. An

exemplary solubility modulator is tartaric acid.

Like the amounts and types of excipients, the amounts and specific type of active ingredient in a dosage form may differ depending on various factors. It will be

understood, however, that the total daily usage of the compositions of the present

disclosure will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular

host will depend upon a variety of factors, including for example, the disorder being

treated and the severity of the disorder; the activity of the specific composition

employed; the specific composition employed; the age, body weight, general health,

sex, and diet of the host; the time of administration; the route of administration; the

rate of excretion of the specific compound employed; the duration of the treatment;

the existence of other drugs used in combination or coincidental with the specific composition employed; and like factors well known in the medical arts. For example,

it is well within the skill of the art to start doses of the composition at levels lower

than those required to achieve the desired therapeutic effect and to gradually increase

the dosage until the desired effect is achieved.

Compositions of the present disclosure are preferably formulated in dosage

unit form for ease of administration and uniformity of dosage. "Dosage unit form" as

used herein refers to a physically discrete unit of the composition appropriate for the

host to be treated. Each dosage should contain the quantity of composition calculated to produce the desired therapeutic affect either as such, or in association with the

selected pharmaceutical carrier medium.

Preferred unit dosage formulations are those containing a daily dose or unit,

daily sub-dose, or an appropriate fraction thereof, of the administered ingredient. For

example, approximately 8 milligrams/kilogram per day of a garlic extract compound

(e.g., allicin) can reduce the mass of adipose tissue in a rat. These results can be used

to predict an approximate amount of the garlic extract compound to be administered to a human or other host, such as cattle.

The approximation includes host factors such as surface area, weight,

metabolism, tissue distribution, absorption rate, and excretion rate, for example.

Therefore, approximately 1 to 100 milligrams/kilogram per day of the ajoene should

produce similar results in humans. As stated above, a therapeutically effective dose

level will depend on many factors, as described above. In addition, it is well within

the skill of the art to start doses of the composition at relatively low levels, and

increase the dosage until the desired effect is achieved.

Pharmaceutical compositions of the disclosure that are suitable for oral

administration can be presented as discrete dosage forms, such as, but not limited to,

tablets (including without limitation scored or coated tablets), pills, caplets, capsules,

chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids,

(such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous

liquid, a non-aqueous liquid, an oil-in- water emulsion, or a water-in-oil emulsion).

Such compositions contain a predetermined amount of the pharmaceutically

acceptable salt of the disclosed compounds, and may be prepared by methods of

pharmacy well known to those skilled in the art. See generally, Remington's

Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton, Pa. (1990). Typical oral dosage forms of the compositions of the disclosure are prepared

by combining the pharmaceutically acceptable salt of disclosed compounds in an

intimate admixture with at least one excipient according to conventional

pharmaceutical compounding techniques. Excipients can take a wide variety of forms, depending on the form of the composition desired for administration. For

example, excipients suitable for use in oral liquid or aerosol dosage forms include, but

are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches,

sugars, microcrystalline cellulose, kaolin, diluents, granulating agents, lubricants,

binders, and disintegrating agents.

Due to their ease of administration, tablets and capsules represent the most

advantageous solid oral dosage unit forms, in which case solid pharmaceutical

excipients are used. If desired, tablets can be coated by standard aqueous or nonaqueous techniques. These dosage forms can be prepared by any of the methods of

pharmacy, hi general, pharmaceutical compositions and dosage forms are prepared by

uniformly and intimately admixing the active ingredient(s) with liquid carriers, finely

divided solid carriers, or both, and then shaping the product into the desired

presentation if necessary.

For example, a tablet can be prepared by compression or molding.

Compressed tablets can be prepared by compressing, in a suitable machine, the active

ingredient(s) in a free- flowing form, such as a powder or granules, optionally mixed

with one or more excipients. Molded tablets can be made by molding, in a suitable

machine, a mixture of the powdered compound moistened with an inert liquid diluent. Examples of excipients that can be used in oral dosage forms of the disclosure

include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders

suitable for use in pharmaceutical compositions and dosage forms include, but are not

limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic

gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose

acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

Suitable forms of microcrystalline cellulose include, but are not limited to, the

materials sold as AVICEL-PH_r101, AVICEL-PH- 103, AVICEL RC-581, and

AVICEL-PH- 105 (available from FMC Corporation, American Viscose Division,

Avicel Sales, Marcus Hook, Pa., U.S.A.), and mixtures thereof. An exemplary

suitable binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or low moisture excipients or

additives include AVICEL-PH- 103™ and Starch 1500 LM.

Examples of fillers suitable for use in the pharmaceutical compositions and

dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate

(e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates,

kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures

thereof. The binder or filler in pharmaceutical compositions of the disclosure typically

accounts for from about 50 to 99 weight percent of the pharmaceutical composition or

dosage form.

Disintegrants may be used in the compositions of the disclosure to provide

tablets that disintegrate when exposed to an aqueous environment. Tablets that contain too much disintegrant may swell, crack, or disintegrate in storage, while those

that contain too little may be insufficient for disintegration to occur and may thus alter

the rate and extent of release of the active ingredient(s) from the dosage form. Thus, a

sufficient amount of disintegrant that is neither too little nor too much to detrimentally

alter the release of the active ingredient(s) should be used to form solid oral dosage

forms of the disclosure. The amount of disintegrant used varies, based upon the type

of formulation and mode of administration, and is readily discernible to those of ordinary skill in the art. Typical pharmaceutical compositions comprise from about 0.5 to 15 weight percent of disintegrant, or from about 1 to 5 weight percent of

disintegrant.

Disintegrants that can be used to form pharmaceutical compositions and

dosage forms of the disclosure include, but are not limited to, agar-agar,, alginic acid,

calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone,

polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches,

pre-gelatinized starch, clays, other algins, other celluloses, gums, and mixtures thereof.

Lubricants that can be used to form pharmaceutical compositions and dosage

forms of the disclosure include, but are not limited to, calcium stearate, magnesium

stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene

glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable

oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and

soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof.

Additional lubricants include, for example, a syloid silica gel (AEROSIL 200,

manufactured by W. R. Grace Co. of Baltimore, Md.), a coagulated aerosol of

synthetic silica (marketed by Degussa Co. of Piano, Tex.), CAB-O-SDL (a pyrogenic silicon dioxide product sold by Cabot Co. of Boston, Mass.), and mixtures thereof. If

used at all, lubricants are typically used in an amount of less than about 1 weight

percent of the pharmaceutical compositions or dosage forms into which they are

incorporated.

This disclosure further encompasses lactose-free pharmaceutical compositions

and dosage forms, wherein such compositions preferably contain little, if any, lactose

or other mono- or disaccharides. As used herein, the term "lactose-free" means that the amount of lactose present, if any, is insufficient to substantially increase the

degradation rate of an active ingredient.

Lactose-free compositions of the disclosure can comprise excipients that are

well known in the art and are listed in the USP (XXI)/NF (XVI), which is

incorporated herein by reference. In general, lactose-free compositions comprise a

pharmaceutically acceptable salt of a catecholamine compound, a binder/filler, and a

lubricant in pharmaceutically compatible and pharmaceutically acceptable amounts.

Preferred lactose-free dosage forms comprise a pharmaceutically acceptable salt of the

disclosed compounds, microcrystalline cellulose, pre-gelatinized starch, and

magnesium stearate.

Since water can facilitate the degradation of some compounds, this disclosure

further encompasses anhydrous pharmaceutical compositions and dosage forms of the

disclosed compounds as active ingredients. For example, the addition of water (e.g.,

5%) is widely accepted in the pharmaceutical arts as a means of simulating long-term

storage in order to determine characteristics such as shelf life or the stability of

formulations over time. See, e.g., Jens T. Carstensen, Drug Stability: Principles &

Practice, 379-80 (2nd ed., Marcel Dekker, NY, N. Y.: 1995). Water and heat

accelerate the decomposition of some compounds. Thus, the effect of water on a formulation can be of great significance since moisture and/or humidity are commonly

encountered during manufacture, handling, packaging, storage, shipment, and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms of the disclosure

can be prepared using anhydrous or low moisture containing ingredients and low-

moisture or low-humidity conditions. Pharmaceutical compositions and dosage forms

that comprise lactose and at least one active ingredient that comprises a primary or secondary amine are preferably anhydrous if substantial contact with moisture and/or

humidity during manufacturing, packaging, and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and stored such

that its anhydrous nature is maintained. Accordingly, anhydrous compositions are

preferably packaged using materials known to prevent exposure to water such that

they can be included in suitable formulary kits. Examples of suitable packaging

include, but are not limited to, hermetically sealed foils, plastics, unit dose containers

(e.g., vials) with or without desiccants, blister packs, and strip packs.

Pharmaceutically acceptable salts of the disclosed compounds can be administered by controlled- or delayed-release means. Controlled-release

pharmaceutical products have a common goal of improving drug therapy over that

achieved by their non-controlled release counterparts. Ideally, the use of an optimally

designed controlled-release preparation in medical treatment is characterized by a

minimum of drug substance being employed to cure or control the condition in an

appropriate amount of time. Advantages of controlled-release formulations include: 1)

extended activity of the drug; 2) reduced dosage frequency; 3) increased patient

compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects;

6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug

activity; and 10) improvement in speed of control of diseases or conditions. See, e.g.,

Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

Conventional dosage forms generally provide rapid or immediate drug release

from the formulation. Depending on the pharmacology and pharmacokinetics of the

drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations

can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like.

Advantageously, controlled-release formulations can be used to control a drug's onset

of action, duration of action, plasma levels within the therapeutic window, and peak

blood levels, hi particular, controlled- or extended-release dosage forms or

formulations can be used to ensure that the maximum effectiveness of a drug is

achieved while minimizing potential adverse effects and safety concerns, which can

occur both from under dosing a drug (i.e., going below the minimum therapeutic

levels) as well as from exceeding the toxicity level for the drug.

Most controlled-release formulations are designed to initially release an

amount of drug (active ingredient) that promptly produces the desired therapeutic

effect, and gradually and continually release other amounts of drug to maintain this

level of therapeutic or prophylactic effect over an extended period of time. In order to

maintain a relatively constant level of drug in the body, the drug must be released

from the dosage form at a rate such that newly released drug will replace the amount

of drug being metabolized and excreted from the body. Controlled-release of an active

ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological

conditions or compounds.

A variety of known controlled- or extended-release dosage forms,

formulations, and devices can be adapted for use with the salts and compositions of

the disclosure. Examples include, but are not limited to, those described in U.S. Pat.

Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595;

5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 Bl; each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for

example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable

membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View,

Calif. USA)), multilayer coatings, microparticles, liposomes, or microspheres, or a

combination thereof to provide the desired release profile in varying proportions.

Additionally, ion exchange materials can be used to prepare immobilized, adsorbed

salt forms of the disclosed compounds and thus effect controlled delivery of the drug.

Examples of specific anion exchangers include, but are not limited to, Duolite® A568 and Duolite® AP 143 (Rohm & Haas, Spring House, Pa. USA).

One embodiment of the disclosure encompasses a unit dosage form which

comprises a pharmaceutically acceptable salt of the disclosed compounds (e.g., a

sodium, potassium, or lithium salt), or a polymorph, solvate, hydrate, dehydrate, co-

crystal, anhydrous, or amorphous form thereof, and one or more pharmaceutically

acceptable excipients or diluents, wherein the pharmaceutical composition or dosage

form is formulated for controlled-release. Specific dosage forms utilize an osmotic

drug delivery system. A particular and well-known osmotic drug delivery system is referred to as

OROS® (Alza Corporation, Mountain View, Calif. USA). This technology can

readily be adapted for the delivery of compounds and compositions of the disclosure.

Various aspects of the technology are disclosed in U.S. Pat. Nos. 6,375,978 Bl;

6,368,626 Bl; 6,342,249 Bl; 6,333,050 B2; 6,287,295 Bl; 6,283,953 Bl; 6,270,787

Bl; 6,245,357 Bl; and 6,132,420; each of which is incorporated herein by reference.

Specific adaptations of OROS® that can be used to administer compounds and compositions of the disclosure include, but are not limited to, the OROS® Push- Pull™, Delayed Push-Pull™, Multi-Layer Push-Pull™, and Push-Stick™ Systems, all

of which are well known. See, e.g. worldwide website alza.com. Additional OROS®

systems that can be used for the controlled oral delivery of compounds and

compositions of the disclosure include, but are not limited to, OROS®-CT and L-

OROS® (see, Delivery Times, vol. 11, issue II (Alza Corporation)).

Conventional OROS® oral dosage forms are made by compressing a drug

powder into a hard tablet, coating the tablet with cellulose derivatives to form a semi¬ permeable membrane, and then drilling an orifice in the coating (e.g., with a laser).

Kim, Cherng-ju, Controlled Release Dosage Form Design, 231-238 (Technomic

Publishing, Lancaster, Pa.: 2000). The advantage of such dosage forms is that the

delivery rate of the drug is not influenced by physiological or experimental conditions.

Even a drug with a pH-dependent solubility can be delivered at a constant rate

regardless of the pH of the delivery medium. However, because these advantages are

provided by a build-up of osmotic pressure within the dosage form after

administration, conventional OROS® drug delivery systems cannot be used to

effectively deliver drugs with low water solubility. This disclosure encompass the incorporation of the compounds of the present disclosure and salts thereof, non-salt

isomers and isomeric mixtures thereof and the like into OROS® dosage forms.

A specific dosage form of the compositions of the disclosure includes at least

the following: a wall defining a cavity, the wall having an exit orifice formed or

formable therein and at least a portion of the wall being semipermeable; an

expandable layer located within the cavity remote from the exit orifice and in fluid

communication with the semipermeable portion of the wall; a dry or substantially dry state drug layer located within the cavity adjacent the exit orifice and in direct or indirect contacting relationship with the expandable layer; and a flow-promoting layer

interposed between the inner surface of the wall and at least the external surface of the

drug layer located within the cavity, wherein the drug layer includes the compound of

the disclosure, a salt thereof, or a polymorph, solvate, hydrate, dehydrate, co-crystal,

anhydrous, or amorphous form thereof. See U.S. Pat. No. 6,368,626, the entirety of

which is incorporated herein by reference.

Another specific dosage form of the disclosure includes at least the following: a wall defining a cavity, the wall having an exit orifice formed or formable therein and

at least a portion of the wall being semipermeable; an expandable layer located within

the cavity remote from the exit orifice and in fluid communication with the

semipermeable portion of the wall; a drug layer located within the cavity adjacent the

exit orifice and in direct or indirect contacting relationship with the expandable layer,

the drug layer comprising a liquid, active-agent formulation absorbed in porous

particles adapted to resist compaction forces sufficient to form a compacted drug layer

without significant exudation of the liquid, active-agent formulation, wherein the

active agent formulation includes the compound of the disclosure, a salt thereof, or a

polymorph, solvate, hydrate, dehydrate, co-crystal, anhydrous, or amorphous form thereof. The dosage form also optionally has a placebo layer between the exit orifice

and the drug layer. {See U.S. Pat. No. 6,342,249, the entirety of which is incorporated herein by reference.)

Parenteral dosage forms can be administered to patients by various routes,

including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intra-arterial. Since administration of parenteral dosage forms

typically bypasses the host's natural defenses against contaminants, parenteral dosage

forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions

ready for injection, dry products ready to be dissolved or suspended in a

pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and

emulsions. In addition, controlled-release parenteral dosage forms can be prepared for

administration to a patient, including, but not limited to, administration DUROS®-

type dosage forms, and dose-dumping.

Suitable vehicles that can be used to provide parenteral dosage forms of the

disclosure are well known to those skilled in the art. Examples include, without

limitation: sterile water; water for injection USP; saline solution; glucose solution;

aqueous vehicles such as, but not limited to, sodium chloride injection, Ringer's

injection, dextrose injection, dextrose and sodium chloride injection, and lactated

Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol,

polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not

limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl

myristate, benzyl benzoate, and mixtures thereof.

Topical dosage forms of the disclosure include, but are not limited to, creams,

lotions, ointments, gels, shampoos, sprays, aerosols, solutions, emulsions, and other forms know to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences,

18th ed., Mack Publishing, Easton, Pa. (1990); and Introduction to Pharmaceutical

Dosage Forms, 4th ed., Lea & Febiger, Philadelphia, Pa. (1985). For non-sprayable

topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one

or more excipients compatible with topical application and having a dynamic viscosity

preferably greater than water are typically employed. Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders,

liniments, salves, and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing

various properties, such as, for example, osmotic pressure. Other suitable topical

dosage forms include sprayable aerosol preparations wherein the active ingredient,

preferably in combination with a solid or liquid inert carrier, is packaged in a mixture

with a pressurized volatile (e.g., a gaseous propellant, such as Frean®), or in a squeeze

bottle. Moisturizers or humectants can also be added to pharmaceutical compositions

and dosage forms if desired. Fragrance and/or other cosmetic ingredients (such as

tint, light reflectors, firming agents, and the like) can also be added to topical dosage

forms, if desired. Examples of such additional ingredients are well known in the art.

See, e.g., Remington's Pharmaceutical Sciences, 18.sup.th Ed., Mack Publishing,

Easton, Pa. (1990).

Transdermal and mucosal dosage forms of the compositions of the disclosure

include, but are not limited to, ophthalmic solutions, patches, sprays, aerosols, creams,

lotions, suppositories, ointments, gels, solutions, emulsions, suspensions, or other

forms known to one of skill in the art. See, e.g., Remington's Pharmaceutical

Sciences, 18th Ed., Mack Publishing, Easton, Pa. (1990); and Introduction to

Pharmaceutical Dosage Forms, 4th Ed., Lea & Febiger, Philadelphia, Pa. (1985). Dosage forms suitable for treating mucosal tissues within the oral cavity can be

formulated as mouthwashes, as oral gels, or as buccal patches. Additional transdermal

dosage forms include "reservoir type" or "matrix type" patches, which can be applied'

to the skin and worn for a specific period of time to permit the penetration of a desired amount of active ingredient.

Examples of transdermal dosage forms and methods of administration that can

be used to administer the active ingredient(s) of the disclosure include, but are not limited to, those disclosed in U.S. Pat. Nos.: 4,624,665; 4,655,767; 4,687,481;

4,797,284; 4,810,499; 4,834,978; 4,877,618; 4,880,633; 4,917,895; 4,927,687; 4,956,171; 5,035,894; 5,091,186; 5,163,899; 5,232,702; 5,234,690; 5,273,755;

5,273,756; 5,308,625; 5,356,632; 5,358,715; 5,372,579; 5,421,816; 5,466;465;

5,494,680; 5,505,958; 5,554,381; 5,560,922; 5,585,111; 5,656,285; 5,667,798;

5,698,217; 5,741,511; 5,747,783; 5,770,219; 5,814,599; 5,817,332; 5,833,647;

5,879,322; and 5,906,830, each of which are incorporated herein by reference in their

entirety.

Suitable excipients (e.g., carriers and diluents) and other materials that can be

used to provide transdermal and mucosal dosage forms encompassed by this

disclosure are well known to those skilled in the pharmaceutical arts, and depend on

the particular tissue or organ to which a given pharmaceutical composition or dosage

form will be applied. With that fact in mind, typical excipients used in form dosage

forms that are non-toxic and pharmaceutically acceptable include, but are not limited

to water, acetone, ethanol, ethylene glycol, propylene glycol, butane- 1, 3 -diol,

isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof.

Depending on the specific tissue to be treated, additional components may be

used prior to, in conjunction with, or subsequent to treatment with pharmaceutically acceptable salts of the compounds of the disclosure. For example, penetration

enhancers can be used to assist in delivering the active ingredients to or across the

tissue. Suitable penetration enhancers include, but are not limited to: acetone; various

alcohols such as ethanol, oleyl, an tetrahydrofuryl; alkyl sulfoxides such as dimethyl

sulfoxide; dimethyl acetamide; dimethyl formamide; polyethylene glycol;

pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone);

urea; and various water-soluble or insoluble sugar esters such as TWEEN 80 (polysorbate 80) and SPAN 60 (sorbitan monostearate).

The pH of a pharmaceutical composition or dosage form, or of the tissue to which the pharmaceutical composition or dosage form is applied, may also be

adjusted to improve delivery of the active ingredient(s). Similarly, the polarity of a

solvent carrier, its ionic strength, or tonicity can be adjusted to improve delivery.

Compounds such as stearates can also be added to pharmaceutical compositions or

dosage forms to advantageously alter the hydrophilicity or lipophilicity of the active

ingredient(s) so as to improve delivery, hi this regard, stearates can serve as a lipid

vehicle for the formulation, as an emulsifying agent or surfactant, and as a delivery- enhancing or penetration-enhancing agent. Different hydrates, dehydrates, co-

crystals, solvates, polymorphs, anhydrous, or amorphous forms of the

pharmaceutically acceptable salt of the compounds can be used to further adjust the

properties of the resulting composition.

Typically, the active ingredient(s) of the pharmaceutical compositions of the

disclosure are preferably not administered to a patient at the same time or by the same

route of administration. This disclosure therefore encompasses kits which, when used

by the medical practitioner, can simplify the administration of appropriate amounts of

active ingredients to a patient. A typical kit includes a unit dosage form of a pharmaceutically acceptable salt

of the compound. In particular, the pharmaceutically acceptable salt of the compound

is the sodium, lithium, or potassium salt, or a polymorph, solvate, hydrate, dehydrate,

co-crystal, anhydrous, or amorphous form thereof. A kit may further include_^a device

that can be used to administer the active ingredient. Examples of such devices

include, but are not limited to, syringes, drip bags, patches, and inhalers.

Kits of the disclosure can further include pharmaceutically acceptable vehicles that can be used to administer one or more active ingredients. For example, if an active ingredient is provided in a solid form that must be reconstituted for parenteral

administration, the kit can include a sealed container of a suitable vehicle in which the

active ingredient can be dissolved to form a particulate-free sterile solution that is

suitable for parenteral administration. Examples of pharmaceutically acceptable

vehicles include, but are not limited to: Water for Injection USP; aqueous vehicles

such as, but not limited to, sodium chloride injection, Ringer's injection, dextrose

injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol,

and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil,

cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl

benzoate.

Having summarized embodiments, reference will now be made in detail to the

illustrative Example. While the disclosure is described in connection with the

Example, there is no intent to limit the embodiments of the disclosure to the following

example. On the contrary, the intent is to cover all alternatives, modifications, and

equivalents included within the spirit and scope of the disclosure. EXAMPLE

This example demonstrates that ajoene-induced ROS generation plays a

central role in the events of the apoptotic pathway and is responsible for activation of

the MAPK pathways. Furthermore, the results indicate that garlic extract compounds such as ajoene play an important role in the translocation of AIF from the

mitochondria to the nucleus, and the results demonstrate the role of PARP in

regulating this process. Materials and Methods:

Chemicals: Phosphate-buffered saline (PBS) and DMEM medium were purchased from GIBCO (BRL Life Technologies, Grand Island, NY). Ajoene was

received as a gift from Rafael Apitz-Castro, Institute for Thrombosis Research,

Caracao, Venezuela. ApoStrand™ ELISA Apoptosis Detection Kit was purchased

from BIOMOL (Plymouth Meeting, PA). The viability assay kit (CellTiter 96

Aqueous One Solution Cell Proliferation Assay) and Caspase-Glo™ 3/7 assay kit

were purchased from Promega (Madison, WI). β-Actin, Catalase and N-acetyl-_L-

cysteine (NAC) were purchased from Sigma (St. Louis, MO, USA). Antibodies

specific for polyclonal anti-AIF, caspase-3, and PARP-I were from Santa Cruz

Biotechnology (Santa Cruz, CA). Antibodies specific for polyclonal anti- phospho-

p38 (Thr¹⁸⁰/Tyr¹⁸²), total p38, phospho-JNK (Thr¹⁸³/Tyr¹⁸⁵), total JNK, phospho-

ERKl/2 (ThT²⁰²ZTyT²⁰⁴), and total ERK1/2 were from Cell Signaling Technology

(Beverly, MA).

Cell line and Cell culture: 3T3-L1 mouse embryo fibroblasts were obtained

from American Type Culture Collection (Manassas, VA) and cultured as described

elsewhere (20). Briefly, cells were cultured in Dulbecco's modified Eagle's medium

(DMEM) containing 10% bovine calf serum (BCS) until confluent. Two days after postconfluence (DO), the cells were" stimulated to differentiate with DMEM containing

10% fetal bovine serum (FBS), 167 nM insulin, 0.5 μM isobutylmethylxanthine

(IBMX), and 1 μM dexamethasone for two days (D2). Cells were then maintained in

10% FBS/DMEM medium with 167 nM insulin for another two days (D4), followed

by culturing with 10% FBS/DMEM medium for an additional 4 days (DS), at which

time more than 90% of cells were mature adipocytes with accumulated fat droplets.

All media contained 100 U/ml of penicillin, 100 μg/ml of streptomycin, and of 292

μg/ml glutamine (Livitrogen, Carlsbad, CA). Cells were maintained at 37°C in a

humidified 5% CO₂ atmosphere.

MTS cell viability assay: Tests were performed in 96-well plates. For mature

adipocytes, cells were seeded (5,000 cells/well) and grown to maturation as described

above. Adipocytes were incubated with either DMSO or increasing concentrations of

ajoene for 6, 12, and 24 h. The medium was then changed and replaced with 100 μl

fresh 10% FBS/DMEM medium and 20 μl MTS solution. Cells were then returned to

the incubator for an additional two hours before 25 μl of 10% SDS was added to stop

the reaction. The absorbance was measured at 490 nm in a plate reader (μQuant™

Bio-Tek Instruments, Inc. Winooski, VT) to determine the formazan concentration,

which is proportional to the number of live cells.

Apoptosis assays: For the assessment of apoptosis, we used the ApoStrand™

ELISA Apoptosis Detection Kit (Biomol, Plymouth Meeting, PA) and Caspase-Glo™

3/7 assay kit (Promega, Madison, WI). The ApoStrand™ ELISA Apoptosis Detection

Kit detects single stranded DNA, which occurs in apoptotic cells but not in necrotic

cells or in cells with DNA breaks in the absence of apoptosis (21, 22). Tests were

performed in 96-well plates. For mature adipocytes, cells were seeded (5,000 cells/well) and grown to maturation as described above. For ssDNA ELISA₅

adipocytes were incubated with either DMSO or increasing concentrations of ajoene

for 3, 6, 12 and 24 h. Thereafter, cells were fixed for 30 min and assayed according to

the manufacturer's instructions. For the caspase activity assay, cells were incubated

with either DMSO (carrier; 0.1%) or increasing concentrations of ajoene for 1, 3, 6,

12 and 24 h. Caspase-3/7 activity was measured using the substrate DEVD- aminoluciferin from the Caspase-Glo™ 3/7 assay kit according to the manufacturer's

instructions.

Measurement of intracellular ROS generation: The determination of ROS was

based on the oxidation of the nonfluorescent 2,7-dichlorodihydroflourescein diacetate

(DCHF) into a fluorescent dye, 2,7- dichloroflourescein "(DCF) by peroxide. Control

cells and cells treated with 200 μM ajoene were analyzed for changes in fluorescence.

Cells were washed twice with PBS and then incubated for 30 min at 37 ⁰C in the dark

with the oxidation-sensitive probe, DCHF (Molecular Probes, Eugene, OR) at 2.5 μM.

Production of ROS was measured by changes in fluorescence at an excitation

wavelength of 495 nm and an emission wavelength of 525 ran.

Western blot analysis: To prepare the whole-cell extract, cells were washed

with PBS and suspended in a lysis buffer (20 mM Tris, pH 7.5, 150 niM NaCl, 1 niM

EDTA, ImM EGTA, 1 % Triton X-IOO, 2.5 mM Na pyrophosphate, ImM β-

glycerophosphate, 1 mM Na₃VO₄, and 100 ug/ml phenylmethylsulfonyl fluoride).

After 30 min of rocking at 4°C, the mixtures were centrifuged (10,00Og) for 10 min,

and the supernatants were collected as the whole-cell extracts. The cytosolic protein

concentration was determined by the method of Bradford (23) with bovine serum

albumin as the standard. Western blot analysis was performed using the commercial NUPAGE system (Novex/Invitrogen), where a lithium dodecyl sulfate (LDS) sample

buffer (Tris/glycerol buffer, pH 8.5) was mixed with fresh dithiothreitol and added to

samples. Samples were then heated to 70°C for 10 min. All cell lysates were

separated by 12% acrylamide gels and transferred to PVDF membranes. The

membranes were blocked with 5% nonfat dry milk in Tris-buffered saline and then

incubated with primary antibodies overnight at 4⁰C. After washing the membranes,

an alkaline-phosphatase-conjugated secondary antibody was added. The target proteins became visible following the addition of 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT), a substrate of alkaline phosphatase.

All experiments were repeated at least two times. Representative Western blots are

shown along with the graphs of the quantitative data.

Quantitative analysis of Western blot data: Measurement of signal intensity on

PVDF membranes after Western blotting with various antibodies was performed using

a FluorChem™densitomer with the AlphaEaseFC™ image processing and analysis

software (Alpha Innotech Corporation). In FIG. 4 density values for the protein bands

of interest are expressed as percentage of the control or 0 h. In FIGS. 5 A and B and 6A, density values are expressed as percent of the highest value in order to show more

clearly how the levels of each protein change. In FIG. 6B density values are expressed

as percent of control. AU figures showing quantitative analysis include data from at

least two independent experiments.

Preparation of nuclear and mitochondrial fractions for measurement of AIF

by Western blot: The cells were washed with ice-cold PBS, left on ice for 10 minutes,

and then resuspended in isotonic homogenization buffer (250 mM sucrose, 10 mM

KCl, 1.5 mM MgCl₂, 1 mM Na-ethyleneglycotetraacetic acid [EGTA], 1 mM NaOH

(Na)-ethylenediaminetetraacetic acid [EDTA], 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonylfluoride, 10 mM HEPES-KOH, pH 7.4). After 80 strokes in a

Dounce homogenizer, the unbroken cells were spun down at 30g for 5 minutes. The

nuclei and heavy mitochondria fractions were fractionated at 75Og for 10 minutes and

14,00Og for 20 minutes, respectively, from the supernatant. The nuclei fraction was

washed 3 times with homogenization buffer containing 0.01% NP-40. AIF in

mitochondrial and nuclear fractions was measured by Western blot as described above.

Detection of DNA fragmentation by gel electrophoresis: Cell pellets (3 x 10⁶

cells) were resuspended in 500 μl of lysis buffer (0.5% Triton X-100, 10 mM EDTA,

and 10 mM Tris-HCl, pH 8.0) at room temperature for 15 min and centrifuged at

16,00Og for 10 min. DNA was then extracted twice with phenolxhloroform (1:1),

precipitated with ethanol, and resuspended in Tris/EDTA buffer (10 mM Tris-HCl,

pH 8.0, and 1 mM EDTA). DNA was analyzed after separation by gel electrophoresis

(2% agarose).

Statistical analysis: Analysis of variance (GLM procedure, Statistica, verion 6.1; StatSoft, Inc.) was used to determine significance of treatment effects and

interactions. Fisher's post-hoc least significant difference test was used to determine

significance of differences among means. Statistically significant differences are

defined at the 95% confidence interval. Data shown are means ± SEM.

RESULTS

Ajoene reduces cell viability: Adipocytes were treated with different

concentrations (0, 50, 100, 200, 400 μM) of ajoene for 6, 12, and 24 h. After

treatment, the number of live cells was determined by MTS assay. As shown in FIG. 1, ajoene time and dose-dependently reduced viability in adipocytes. Ajoene at 200

μM decreased cell viability about 50% after 24 h of treatment. These concentrations

of ajoene were selected for subsequent analyses.

Ajoene induces apoptosis: To investigate whether the reduction in cell number by ajoene was due to apoptosis, a ssDNA ELISA assay was used as a determinant of

cellular apoptosis. As shown in FIG. 2A, exposure of adipocytes to ajoene resulted in

a time- and dose-dependent induction of cell death that was detected after 6 h with

200 μM ajoene and higher. Longer (e.g., 24 h) exposures to ajoene significantly

increased apoptosis at 200 and 400 μM ajoene (p<0.05). DNA extracts from

adipocytes treated with ajoene displayed a characteristic apoptotic ladder pattern of

discontinuous DNA fragments on agarose gel electrophoresis (FIG. 2B).

Ajoene triggers apoptosis by oxidative stress: To determine the involvement of

ROS in ajoene-induced apoptosis, ROS levels were determined in ajoene-treated

adipocytes. As shown in FIG. 3A, ajoene increased ROS production by 2.1 fold after

5 min, reaching a plateau after 20 min. There was a decrease in ROS production between 20 and 40 minutes after treatment. Furthermore, when adipocytes were

pretreated with 10 mM NAC (a thiol-containing antioxidant) for 1 hr prior to

incubation with ajoene, ajoene-induced apoptosis was effectively reduced (FIG. 3B) to

nearly the control level. However, pretreatment of adipocytes with 400 units of

catalase (a scavenger for hydrogen peroxide) did not prevent the ajoene-induced

apoptosis.

Ajoene induces activation of MAPKs: Since the apoptotic effects of ajoene

may involve the generation of ROS (8) and some alterations in oxidative state are

intimately connected to perturbations in MAP kinase pathways (9), studies were

undertaken to determine the relationship between ajoene-induced oxidative stress and changes in JlSfK and ERKl/2 activation. Incubation of adipocytes with ajoene (200

μM) led to phosphorylation of JNK (FIG. 4A, upper panel). Quantitative analysis

(FIG. 4A) shows that activation of JNK occurred as late as 60 min with maximum

activity at 180 min, after which JNK decreased gradually. Interestingly, ERKl/2, a

kinase suggested to play a role in survival pathways, was also phosphorylated upon

exposure to ajoene. FIG. 4B (upper panel) shows the time-dependent phosphorylation

of ERKl/2 in adipocytes treated with ajoene (200 μM). Maximum activation of

ERKl/2 was observed 30 min after incubation with ajoene and quickly diminished

after 60 min. Pretreatment with the antioxidant NAC (10 niM) completely blocked

both ajoene-mediated ERKl/2 activation and JNK activation (FIGS. 4C and 4D).

Ajoene induces PARP-J cleavage and AIF-mediated cell death in a caspase-

independent manner: Since it has been shown that ROS-mediated DNA damage

triggers activation of PARP and subsequent cell death (18, 25), the role of PARP in

ajoene-induced apoptotic cell death was also investigated. Treatment of adipocytes

with 200 μM ajoene induced the proteolytic cleavage of PARP-I (116 kDa), resulting

in the accumulation of the 85 kDa cleavage product (FIG. 5 A, upper panel). Western

blotting and quantitative data revealed that PARP-I cleavage was apparent 4 hr after ajoene treatment and gradually increased. Moreover, pretreatment with the

antioxidant NAC blocked ajoene-mediated degradation of PARP-I (FIG. 5B). Surprisingly, ajoene did not activate caspase-3; either through proteolytic cleavage of

the proenzyme (FIG. 5A) or through protease activity (FIG. 5C). Taken together,

these data indicate that ROS-mediated cell death involved PARP degradation in a

caspase-independent manner.

Whether ajoene-induced cell death involved apoptosis-inducing factor (AIF)

was examined, because AJP-induced cell death is also caspase independent (17). The adipocytes exposed to ajoene (200 μM) showed a translocation of AIF from the

mitochondria to the nuclei in a time-dependent manner (FIG. 6A). Quantitative

analysis also showed that the amount of nuclear AIF gradually increased from 4 h to

12 h. Moreover, the pretreatment with the antioxidant NAC prevented translocation

of AIF to nuclei (FIG. 6B).

DISCUSSION

The present example elucidates the biological effect of ajoene, a component of

garlic, on 3T3-L1 adipocytes. These results represent the first insight into the mechanism of ajoene-induced apoptosis by ROS in 3T3-L1 adipocytes. ROS generation is first shown here to activate the MAPK cascade and PARP-I . These

results show that increased ROS production is involved in AIF release in a caspase-

independent manner.

To elucidate the mechanism of apoptosis by ajoene, MAPK expression and

ROS production were investigated. Results show that treatment with ajoene led to

phosphorylation of both ERK1/2 and JNK in 3T3-L1 adipocytes. In general, JNK

activation is associated with apoptosis induction, whereas ERK activation is

cytoprotective (14). Although it is generally accepted that activation of ERKl/2 leads

to cell proliferation (26), there are conditions in which ERKl/2 activation results in cell death (27, 28). Furthermore, previous studies have shown that H₂O₂ (ROS

stimulator) rapidly activated ERK in PC 12 cells (29), and ROS are involved in cell

death via the ERKl/2 signaling pathway (30). The present results also showed that

ROS levels peaked 20 min after ajoene treatment, whereas ERKl/2 was activated at 30 min and JNK was activated 180 min, which suggests that within this context, ROS

is likely to be upstream of MAPK activation. These data indicate that ajoene

differentially influences the phosphorylation status of members of the MAP kinase superfamily; the phosphorylation of ERK1/2 rapidly decreased, whereas the

phosphorylation of INK slowly and modestly decreased. In addition, the observation

that pre-treatment of cells with NAC inhibited both ROS generation and ERK1/2 and

JNK protein expression suggests that ROS directly influences MAPK signaling.

Catalase is a scavenger for hydrogen peroxide and did not block ajoene-induced ROS generation. NAC, however, is a thiol-reducing agent in addition to its action as a free

radical scavenger (31). Ajoene, which contains thiol groups, may be reduced by NAC and thereby lose its ability to induce apoptosis through generation of ROS and subsequent MAPK activation.

Some studies have implicated the members of the MAPK family as regulators

of mitochondrial-dependent apoptosis (32). A recent report showed that JNK up-

regulation is followed by the activation of Bax and down-stream mitochondrial

depolarization, with the release of AIF and cytochrome c, in a caspase-independent

manner (33). Therefore, these signaling pathways may be involved in the ROS-

induced mitochondrial changes by ajoene. The present results demonstrate that ajoene

did not affect the activity of caspase-3 either by proteolytic cleavage of the proenzyme

or by protease activity. This suggests that the apoptotic cell death induced by ajoene

treatment is caspase independent.

It has been reported that AIF mediates cell death through a caspase-

independent pathway. Death stimuli causes translocation of AJJF from the

mitochondria to the nucleus, where it initiates nuclear condensation (17, 34). Once

the nucleus condenses, this leads to large-scale chromatin fragmentation followed by

cell death. Consistent with these findings, translocation of AlF from mitochondria to

the nucleus and DNA fragmentation occurred. In addition, the treatment of cells with

NAC inhibited AIF translocation to the nucleus, suggesting that the increased intracellular ROS level is critical for the AIF relocalization after ajoene treatment.

These observations indicate that ajoene induces ROS generation followed by AIF

translocation and cell death, which can be reversed by preventing generation of ROS.

PARP is a nuclear enzyme that facilitates DNA repair in response to DNA

damage (35, 36). Enhanced activation of poly(ADP-ribose) polymerase (PARP)

enzyme is a major contributor to oxidative stress-induced cell dysfunction and tissue

injury (37, 38). Reactive oxygen species (ROS) cause single-strand DNA breaks (39).

Single-strand DNA breaks can activate nuclear PARP. IfPARP activation exceeds a certain limit, it can lead to cellular NAD⁺ and ATP depletion, ultimately resulting in

cell death (37-40). The present data also showed that an increased level of ROS by ajoene induced cleavage of 116 kDa PARP-I resulted in the accumulation of an 85

kDa product. Further treatment with NAC attenuated cleavage of PARP-I.

hi summary, these findings demonstrate that ajoene treatment induced

apoptosis through the caspase-independent cell death pathway by enhancement of

intracellular ROS level. The enhancement of intracellular ROS level promotes

MAPK and PARP-I activation and subsequent AIF release, ultimately resulting in

apoptosis. Thus, ajoene may be a new therapeutic tool for the treatment of obesity by regulating fat cell number through the induction of adipocyte apoptosis.

It should be emphasized that the above-described embodiments of the present

disclosure are merely possible examples of implementations, and are set forth only for

a clear understanding of the principles of the disclosure. Many variations and

modifications may be made to the above-described embodiments of the disclosure

without departing substantially from the spirit and principles of the disclosure. All

such modifications and variations are intended to be included herein within the scope

of the present disclosure and protected by the following claims. References:

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Claims

CLAIMS The following is claimed:

1. A method of treating obesity comprising administering to a host in need of

treatment an effective amount of a compound selected from: ajoene,

precursors thereof, and derivatives thereof, wherein the compound is present in

a dosage level effective to initiate the release or activation of an apoptosis

inducing factor, wherein the apoptosis inducing factor leads to the apoptotic cell death of adipose tissue cells in the host.

2. A method for inducing apoptosis in adipose tissue cells in a host, comprising

initiating the release or activation of an apoptosis inducing factor by

administering an effective amount of at least one garlic extract compound,

wherein the apoptosis inducing factor leads to apoptotic cell death.

3. The method of claim 2, wherein the garlic extract compound is selected from

garlic thiosulfmates and transformation products thereof, ajoene, precursors thereof, and derivatives thereof; allicin, precursors thereof, and derivatives

thereof; allyl methanethiosulfinate, precursors thereof, and derivatives thereof;

disulfides, precursors thereof, and derivatives thereof; allylsulfides, precursors

thereof, and derivatives thereof; vinyldithiins, precursors thereof, and

derivatives thereof; diallyl trisulfides, precursors thereof, and derivatives

thereof; and mercaptocysteines, precursors thereof, and derivatives thereof;

and combinations thereof.

4. The method of claim 2, wherein the garlic extract compound is selected from

ajoene, precursors thereof, and derivatives thereof.

5. The method of claim 4, wherein administering an effective amount of at least

one garlic extract compound does not activate caspases.

6. The method of claim 4, wherein the apoptotic cell death occurs independently from caspases.

7. The method of claim 2, wherein the apoptosis inducing factor is Apoptosis

Inducing Factor (AIF).

8. The method of claim 2, wherein the apoptosis inducing factor is a mitogen-

activated protein kinase (MAPK).

9. The method of claim 8, wherein the MAPK is extracellular signal-regulating kinase (ERK1/2).

10. The method of claim 8, wherein the MAPK is Jun-N-terrninal kinase (INK).

11. The method of claim 2, wherein the apoptosis inducing factor is PoIy(ADP

ribose) polymerase (PAPvP).

12. A pharmaceutical composition comprising at least one garlic extract

compound in combination with a pharmaceutically acceptable carrier, wherein the at least one garlic extract compound is present in a dosage level effective

to initiate the release of an apoptosis inducing factor, wherein the apoptosis

inducing factor leads to the apoptotic cell death of adipose tissue cells in a

host.

13. The pharmaceutical composition of claim 12, wherein the apoptotic cell death of adipose tissue cells treats obesity.

14. The pharmaceutical composition of claim 12, wherein the garlic extract

compound is selected from garlic thiosulfinates and transformation products

thereof, ajoene, precursors thereof, and derivatives thereof; allicin, precursors

thereof, and derivatives thereof; allyl methanethiosulfmate, precursors thereof,

and derivatives thereof; disulfides, precursors thereof, and derivatives thereof;

allylsulfides, precursors thereof, and derivatives thereof; vinyldithiins,

precursors thereof, and derivatives thereof; diallyl trisulfides, precursors

thereof, and derivatives thereof; and mercaptocysteines, precursors thereof, and derivatives thereof; and combinations thereof.

15. The pharmaceutical composition of claim 12, wherein the garlic extract

compound is selected from ajoene, precursors thereof, and derivatives thereof.

16. The pharmaceutical composition of claim 12, wherein the garlic extract

compound is selected from garlic thiosulfinates and transformation products thereof.

17. The pharmaceutical composition of claim 12, wherein the garlic extract

compound is selected from allicin, precursors thereof, and derivatives thereof.

18. The pharmaceutical composition of claim 12, wherein the garlic extract

compound is selected from allyl methanethiosulfinate, precursors thereof, and

derivatives thereof.

19. The pharmaceutical composition of claim 12, wherein the garlic extract compound is selected from disulfides, precursors thereof, and derivatives

thereof.

20. The pharmaceutical composition of claim 12, wherein the garlic extract

compound is selected from allylsulfides, precursors thereof, and derivatives

thereof.

21. The pharmaceutical composition of claim 12, wherein the garlic extract

compound is selected from vinyldithiins, precursors thereof, and derivatives

thereof.

22. The pharmaceutical composition of claim 12, wherein the garlic extract

compound is selected from diallyl trisulfides, precursors thereof, and

derivatives thereof.

23. The pharmaceutical composition of claim 12, wherein the garlic extract

compound is selected from mercaptocysteines, precursors thereof, and

derivatives thereof.

24. The pharmaceutical composition of claim 12, wherein the garlic extract

compound is selected from S-allylmercaptocysteine (SAMC), S-

methylcysteine (SMC), S-allyl cysteine sulfoxide (SACS), diallyl disulfide (DADS), S-allylcysteine (SAC), S-ethylcysteine (SEC), S-ρropylcysteine

(SPC), and combinations thereof.

25. The pharmaceutical composition of claim 12, wherein the garlic extract is

selected from ajoene, precursors of thereof, and derivatives thereof, wherein

the dosage level of the ajoene, precursors of thereof, and derivatives thereof,

does not activate caspases.

26. The pharmaceutical composition of claim 12, wherein the apoptotic cell death of adipose tissue cells in the host occurs independently from caspases.

27. A method for treating osteoporosis in a host, comprising administering an

effective amount of at least one garlic extract compound.

28. The method of claim 27, wherein the garlic extract compound is selected from

ajoene, precursors thereof, and derivatives thereof.

29. A pharmaceutical composition comprising at least one garlic extract

compound in combination with a pharmaceutically acceptable carrier, wherein

the at least one garlic extract compound is present in a dosage level effective

to treat osteoporosis.

30. The pharmaceutical composition of claim 29, wherein the garlic extract

compound is selected from ajoene, precursors thereof, and derivatives thereof.