Note: Descriptions are shown in the official language in which they were submitted.
WO 93/12654 21 Z 6 ~5 PCr/US92/10979
MEDI~AL IJSE!; OF TR~OXOLANE AND DIPEROX~DE C()MPOUI~ S
Background of the ~nvention
This invention relates to trioxolane and diperoxide compounds. More particularl~it relates to formation of these compounds from unsaturated hydrocarbons and
pharmaceutical prepara~ions including these compounds for treating or pre~entinP medical
corlditions. lt also relates to methods of treating or preventing medical conditions usin_ the
trioxolane and diperoxide compounds.
A trinxolane compound is herein defined as a compound of the generaJ structure: -~
I~C--R'
\0/ .
wherein R and R' represent the same or different organic moieties. The indicatedcarbons may also have additional organic moiety branches.
A diperoxide compound is herein defined as a compound ~f ~he general structure:
0~)
/ ~ .
R~ C--R'
O{) '
~5 wherein R and R' represent the same or different organic moieties. The indicated carbons
may also have additional organic moiety branches.
Procedures for ozonating oil-soluble compounds are known in the art. bein~
disclosed, for example, in U.S. patent No. 92~,~90 to Neel, U.S. Patent No. ~.083.57 ' to
McKee, and V.S. Patent No. 4,451,480 to De Yillez. However, not all ozonation reactions
result in the production of trioxolane and diperoxide compounds. The production of such
compounds from unsaturated hydrocarbons is disclosed in Murray et al., "Ozonolvsis:
Forma~ion of Cross Diperoxides" and Giegee et al, "Fragmentation of Ozonides t~ -
Solvents," both in Ozone Reactions with t~r~anic Compounds, Advances in Chemistr- Series
112, American Chemical Society, Washington, D.C. (1972). 'rhe disclosures of these tuo
_~ references are incorporated herein in their entirety by reference thereto.
`
~VO ~3/126~4 PCr/US92/1097
-2 -
~, 6 ~ 5 1 Ozonation of olefins is generally recognized in terms of a mechanism postulated by
Criegee, supra. This mechanism provides that ozone reacts with an unsaturated bond to
form an initial, unstable prima~y ozonide (R--C--O3-C--R'). ~is primary ozonide readily
decomposes to form a zwitterion and a carbonyl fragment. ~hese ~ragments can then
S combine to give a trioxolane compound. Under other conditions, the zwitterion may
dimerize to form a diperoxide derivative.
The prior art discloses that some particular types of ozonated chemical compositions
have certain pharmacological activities. However, as far as Applicants can ascertain, none
of these compositions appear to have been prepared in a manner likely to result in the
formation of substantial quantities of diperoxide or trioxolane compounds.
In U.S. Patent No. 925,590, Neel discloses the use of ozonated hydrocarbons for
inhalation therapy, because it was believed to have a therapeutic effect for consumption and
asthma. Even had the ozonation system of Neel resulted in the formation of substantial
quantities of diperoxide or trioxolane compounds, such compounds have very low vapor
pressures. Thus, only insubstantial quantities of diperoxide or trioxolane compounds would
be expected to be found in vapor.
Knox, U.S. Patent No. 1,210,949 disdoses ozonation of castor oi} in order to produce
a laxative. Ozonation of the oil was believed to reduce its toxicity and create a germicidal
effect. In order to produce substantial quanti~ies of diperoxide or trioxolane compounds
using the method disclosed by Knox, temperatures approachin~ -50C using a very dilute
solution would be required.
Johnson, V.S. Patent No. 2,356,06 discloses the use of ozonides of glycerine
trioleates for external application, because it was believed that those particular triglycerides
had a germicidal, fungicidal, and deodorizing effect. The methods of Johnson. for reasons
described above in connection with the patent to Knox, are also not believed to result in the
production of significant quantities of diperoxide or trioxolane compounds.
DeVillez, U.S. Patents Nos. 4,451,480 and 4,591,602, discloses use of ozonides of
certain fatty acids, including olive oil. sesame oil, jojoba oil, castor oil and peanut oil, for
ex~ernal use as antimicrobial agents, particularly in the treatment of acne. It is believed that
a. !east some of these compounds cause unacceptable skin irritation. DeVillez discloses
ozonation at 35-65C, a temperature at which diperoxides and trioxolanes are not expected
to ~e formed in substantial quantities.
Accordingly, so far as can be determined, none of she medical uses of ozona~ed
cc.npounds described in the prior art have ever made use of substan~ial quantities of
21~ 7
~VO 93/~26~4 PCr/US92/1097
-3-
trioxolane or diperoxide compounds. Moreover, none of the prior art ozonated compounds
appears to have ever been commercialized for medical applications. Presumably, ~his lack
of commercialization is due to unaceeptable side-effects, toxicity, difficulties in storage, or
minimal effectiveness. Many of these various compositions decompose on standing.
S Immunomodulation offers an opportunity to treat a variety of medical conditions.
For example, both infections and neoplasms can be treated by increasing the immune
response thereto. Some allergic reactions and other au~o-immune responses can also be
treated through immunomodulation. However, there are few effective immunomodulatonr
therapies known; and many of the known ~nmunomodulatory therapies produce untoward
side effects. Thus, there is a need for safe and effective immunomodulatory treatment.
At any one time, it is estimated that 1/3 of all women are suf~ering from bacterial
or fungal vaginal infection. The only presently available treatments are time consuming and
the medications used are irritating to mucous membranes. Thus. there is a need for a
relatively non-irritating, safe, and effective composition for treatment of these infections.
Genital herpes lesions and Herpes simplex lesions are notoriously resistant to
treatment. These viral infections inflict a significant percensage of the population~ and there
is, at present, no known cure. Thus, a need exists for compositions that can trea~ herpes
lesions in at least a palliative manner to minimize the discomfort suffered by those suffering
from these diseases.
~0 Chicken pox (Herpes wster) is a common childhood disease, for which no vaccine
is currently known. Lesions of chicken pox cause itching, and may lead to permanent
- disfigurement, if scratched. Since the disease strikes mainly children~ who are unable to
resist scratching, the need exists for compositions that can anti-puritically treat chicken pox
lesions to minimize disfigurement caused by the disease.
External fungal infections, such as athletes foot and ~nychomycosis (fungal infections
of the nails), afQict a large portion of the human population. Similar fungal infections afnict
a large percentage of the animal papulation. Current treatments for external fungal
infections are irritating to sensitive indiYiduals, and not always effective. ln addition.
onychomycosis is difficult to treat, and its incidence appears to be on the rise with the
advent of acrylic and other adhesivehy-mounted artificial naDs. Therefore~ a need exists for
a relativeJy non-irritating, effective treatment for these infections.
Jndolesli neoplasms of the skin, such as warts and moles, also afflict a large portion
of the human and animal population. Current over-the-counter medications are not always
effective, and the only effective therapy in some instances ;s to have the neoplasms frozen
WO 93/12~S4 PCr/Uâ92~10
-4-
2 ~ 2 6 ~ ~ ~r burned of ~, necessitating a doctor's visit. Thus~ a need exists for a treatment which is
effective, and which can be applied by the patient or owner of the afflicted animal.
Stervidal medications are currently in widespread use to relieve the discom~orts of
bee stings, insect bites, and other dermatoses, such as those caused by psoriasis, poison oak,
S or poison ivy. While these medications are sometimes effective, their long term use can
result in side effects, including thinning of the skin, sleeplessness, physical deformation,
improper fat deposition, dependency, and others. Thus, there is a need ~or an effective
alternative medication for these ailments.
Symptoms of sunburn can range from mild discomfort to severe burns. This
condition oc~asionally affec~s virtually the entire population. Current treatments do Jittle
more than mask the pain associated with this condition. Products which prevent sunburn,
when applied prior to exposure, are currently available. However, there is no product
currently available which prevents sunburn symptoms or alleviates the severitv of sunburn
when applied after exposure to the sun. Many people carelessly or inad~ertently expose
I5 themselves to Ihe sun without using protective sunscreens. Thus, a need exists for a product
that can preveDt sunburn after exposure to the sun.
In the treatment of severe burns, prevention of dehydration and infection in theburned patient are major concerns. Currently used therapies for severe burns vhich address
these concerns are often irritating to sensitive, burned tissues. Thus, there is a need for a
method of treating burns that is non-irritating, yet still effective against both dehydration
and infection.
Many adolescents and young adults suffer from acne. Many compounds are
currently available to treat acne, with variable effectiveness. The most effective
compositions currently known to treat acne use active oxygen to kill the bacteria which are,
25, in part, respsnsible for the condition. These include benzoyl peroxide. However. these
compositions are sometimes irritating, do not always deliver enouRh oxygen for optimal
effectiveness, and can cause drying of the skin. Thus, a need exists for a non-desiccating,
effective, and non-;rritating treatment for acne.
SexuaLly transmitted dise?ses (STDs), including herpes, syphilis, gonorrhea and
3û AIDS, are endemic in today's society. Condoms are currently the most effective means of
preventing the transmission OI .hese diseases. However, condoms are not 100C~c effective.
A need. therefore, exists for ~;e~arations which increase the effectiveness of condoms in
preventing the transmission uf Sl~s.
WO~3/12654 2126?~ 7 P~/~S9~/J0979
Both topical and systemic Leishmaniasis are widespread throughout the tropical
areas of .he world. Presently, at least 4,000,000 people are hlQW tO be infected with a
parasite which causei, one of these conditions. No totally effective therapies are known.
Accordingly, a clear need is evident for an effective treatment or t}~erapy for ~hese diseases.
S Summary of the Invention
One aspect of the present invention provides a method of medical trealment for acondition, including an oral pharyngeal medical condition, in a mammal. This method
includes the application to the mammal of a pharmacologically effectiYe amount for
treatment of the condition of a trioxolane or a diperoxide of a non-terpene unsasurated
1~ hydrocarbon. For an oral pharyngeal medical condition, the compound is applied in a forrn
sui~able for oral pharyngeal application. Preferably, this form is a mouthwash. throat spray,
oral rinse, troche or medicated chewing gum. E~samples of conditions which can be treated
with this method include sore throat and infection of the oral pharyngeal areas of the
mammal, such as bacterial, viral or fungal infection, including candidiasis. Preferred active
15 ingredien~s include 3-hexene-1-ol and erucic acid.
Another aspect of the present irvention provides a pharmaceutical composition for
the treatment of a medical condition, such as an oral pha~yngeal medical condi~ion, in a
mammal. This composition includes a pharmaceutically effective amount for treatmenl of
the condition of a trioxolane or a diper~xide of a non-terpene unsaturated hydrocarbon in
20 a pharmaceutically acceptable, non-aqueo.)s carrier. ln this embodiment. the composition
is preferably in a form selected from the group consisting of throat spray, oral rinse, troche
and medicated chewing gum.
Another aspect of the present invention provides a method of medical treatment for
a systernic disorder in a mammal. This method includes the application to the intestine of
25, the mammaJ of a pharmacologically effective amount for ~reatment of ~he condition of a
trioxolane or a diperoxide of an unsa~urated hydrocarbon in a form which releases active
ingredient in the intestine for intestinal absorption. The disorder ~reated can be any of a
variety of systemic disorders, in~luding intestinal infection. The hydrocarbon used can be
a terpene or a non-terpene.
Along with the foregoing me~hod of trealing systemic disorders, the present
invention provides additional pharmace~-.cal compositions for the treatment of systemic
disorders in mammaL These compositio~;., include a pharmaceutically effective amount for
trealment of the disorder of a trio~oJane or a diperoxide of an unsatura~ed hydrocarbon in
a pharmaceuticalJy acceptable, non-agueo !s carrier. The form of this composition pro~rides
WO 93/12654 PCr/U~92~10974
-6-
2~æ6~ for release of the trioxolane or diperoxide derivative in the intestine. As noted above in
connection with the method, the hydrocarbon derivative can be of a terpene or non-terpene.
In order to provide for release in the intestine, the composition can be in a form
appropriately coated to prevent diges~ion by stomach enzymes. Preferred forms of the
composi~ion include gel caps, capsules and lozenges.
Another aspect of the present invention provides a method of medical treatment for
a medical condition in a mammal. The method indudes the application to the mammal of
a pha2macologically effective amount for treatment of the condition, of a trioxolane or a
diperoxide of a non-terpene unsaturated hydrocarbon. l he application can advantageously
parenteral, topical or other method known to those of ordinary skill in the art. In cerlain
preferred embodiments, the non-terpene unsaturated hydrocarbon is either 3-hexene-1-ol
or erucic acid.
Other aspects of the present invention provide a method of modulating the immunesystem of a mammal, a method of treating bacterial infections in a mammal, a method of
1~ treating fungal infections in a mammal, a method of trealing protozoal infections in a
mammal, a method of treating viral infections in a mammal, and a methQd of treating
inflammation of a tissue in a mammal. Each of these methods includes applying a
pharmacologically effective amount of a trioxoiane or a diperoxide of a non-terpene
unsaturated hydrocarbon to the mammal in an amount effective to produce the desired
treatment. These methods can also include hydrolyzing the trioxolane or diperoxide
derivative to produce a therapeutic molecule having a therapeutic effect on the mammal.
Preferably, this therapeutic molecule comprises a carbonyl zwitterion. A varie~ of medical
conditions can be treated using these methods. For example, HIV infection~ fungal diseases
of the skin, bacterial diseases of the skin, impetigo, paronychia, viral diseases of the skin
~5 (such as herpes infection, venereal warts, and common warts), dermatological conditions
(such as eczema, psoriasis, insect bites. coral burns, jellyfish stings, poison oak, seborrheic
dermatitis and burns), dental or oral conditions (such as pharyngitis, tooth ache, halitosis.
canker sores and gingivitis), and hemorrhoids can all be treated with these methods. The
non-terpene unsaturated hydrocarbon used in these methods can be any of a number of such
compounds, such as erucicacid or3-hexene-1-ol. The present invention also includes
pharmaceutical compositions for the treatment of a medical condition in a mammal. These
compositions include a pharmaceuticaLly effective amol~nt for treatment of the condi~ion.
of a trioxolane or a diperoxide of a non-terpene unsaturated hydrocarhon in a
pharmaceutically acceptable, non-aqueous carrier. '~ertain preferred forms of these
WO ~3~2654 21 2 6 75 7 PCI/US92~10979 -~
-7-
compositions are toothpaste. mouthwash. rectal or vaginal supposi~ories, topicalpreparations, and forms suitable for sublingual application or parenteral administration.
The composition can also be applied to a condom or be made in combination with soap.
Another aspect of the present invention provides a method of preventing sexual~y
S transmitted infections or preventing pregnancy in a mammal. lllis method includes
application to the genitals of the mammal of a trioxolane or diperoxide derivative of an
unsaturated hydrocarbon.
Still another aspect of the present invention provides a meihod of treating cancer
in a mammal having cancer, comprising the application to the mammal of a
pharmacologically effective amount for treatment of eancer, of a trioxolane or a diperoxid
e
of a non-terpene unsaturated hydrocarbon. In certain preferred embodiments of this
method the method is used to treat a mammal that has a cancer such as adenocarcinoma
of the lung, Hodgkin's Disease, or lymphoma.
The present invention also indudes a method of treating rheumatoid arehritis~
osteoarthritis or inflammatory polyarthritis in a mammal. This method includes identifying
a mammaJ having one of the conditions, and applying a trioxolane or a diperoxide of an
unsa~urated hydrocarbon to the mammal.
Stil~ other aspects of the present invention provide methods of treating topicai or
systemic leishmaniasis, chronic fatigue s3rndrome or lupus e~ythematosus in a mammal.
These methods include identifying a mammal having the indicated cond;tion and applyin~
a trioxolane or a diperoxide of an unsaturated hydrocarbon to the mammal. In a similar
aspect of the invention, there is provided a method of treating a physical wound to a tiss
ue
of a mammal. This aspect of the invention includes identifying a mammal having a wound
to a tissue thereof, and app~ying a trioxolane or a diperoxide of an unsaturated hydrocarb
on
to the tissue. This method can also include preventing scar formation through application
of a trioxolane or a diperoxide of an unsaturated hydrocarbon 10 the tissue.
Other aspects of the present invention will be made clear from the followin~g detaile
d
description.
DetaiJed Description of the Invention
Unsaturated Hvdrocarbon Startin~ Materials
Terpene hydrocarbons are also known as isoprenoids, because they rnav general1y
be constructed from isoprene units (C--C C--C=C). Thus. terpene hydrocarbons areusualJy exact multiples of C5H8. Terpenes are cJassified according to the number of
isoprene units of which they are composed, as shown in Table 1.
WO 93/12654 P~/U~92/1~97~9
-8-
~ 6r~ TABLE ~
1 hemi- 5 ses-
2 mono- 6 tri-
3 sesqui- 8 tetra-
4 di- n poly-
While not limiting the scope of the invention, examples of terpenes which can pr~ve
especially effective, when used in certain preferred me~hods of the presen~ invention inslude
limonene, citrunella, alpha-carotene, beta-carotene, vjtamin A, linalool, linalyl acetate, and
squalene~ Other compounds which are believed to make pharmacologically active terpene
trioxolane or diperoxide derivatives in accordance wi~h the present invention include
geraniol, limonene, alpha-pinene, loganin, c)nnene, farnesanes, eudesmanes. acoranes,
cedranes, chamigranes, caryophyllanes, illudanes, humulenes, himachalenes, longifolanes,
perhydroazulenes, quaianes, quaianolides, and germacranes~ Still other compounds which
are believed to make pharmacologically active terpene trioxolane or diperoxide derivatives
in accordance wi~h the present invention include labdanes, clerodanes, abietic acid,
phyllocladene, giberellins, ophiobolin A, retigeranic acid, gasgardic acid, lanosterol, euphol.
oleanane, ursane, lupeol, hydro~hopanone, lupanes, and hopanes~ Other panicular terpene
compounds whiçh are believed to make pharrnacologically active terpene trioxolane or
diperoxide derivatives when prepared in accordance with the present invention include B-
selinene, zingibene. camphene, sabinene, ocimene, myrcene, nerol. citral A. citral B. farnesoL
bisabolene, phytol, and cecropia honnone~ Trioxo~ane or diperoxide derivatives of terpenes
have three or four oxygen atoms respectively rep!acing the double bonds at sites of
unsaturation, creating a trioxyacyclopentane referred to herein as a trioxolane derivative.
We have now discovered that pharmacologically active compounds can be produced through
szonation of both terpene and non-terpene unsaturated hydrocarbons under conditions
which lead to the formation of substantial quantities of trioxolane or diperoxide derivatives
thereof~
Methnds of ~thesis
ln the preparation of trioxolane and diperoxide derivatives, the particular terpene
or non-terpene unsaturated hydrocarbon starting material is firs~ obtained~ A large and
WO 93/126~4 2 1 2 6 ~ 7 P~/US92/1~979
g
representa~ive number of terpene and non-terpene unsaturated hydrocarbon starting
material compounds are disclosed in the literature and/or are commercially available.
In the trioxolane or diper~xide compound synthesis, ozone is passed through the
unsaturated hydrocarbon starting material under conditions that provide for intimate conta~
S between ~he starting material and the ozone, such as thin film procedures, sparging, gas
entrainment procedures, and the like. On a small scale, for example, the unsaturated
hydrocarbon is placed in a vented ve~sel, and ozone is sparged through ~he material until
the reaction is complete.
Trioxolane derivatives are generally favored in the ozonation of unsaturated
hydrocarbons. However, diperoxide derivatives are genera)ly produced from trans isomers
of asymmetric compounds and ~rom hydroearbon compounds having sites of unsaturation
including a tertiary carbon. Wllen compounds which tend to produce diperoxide derivative~
are used in the ozonation reaction, slightly higher temperatures can be tolerated in order
to produce the diperoxides. Thus, these compounds can be o~onated at temperatures up
to 35C. The ozonation of compounds which tend lo produce trioxolane derivatives should
generally be undertaken at temperatures less than 25C in order to produce substantial
quantities of the derivatives.
It is important to use a proper combination of solvent and tempera~ure in order to
generate substantial quantities of trioxolane or diperoxide derivatives. For generation of
substantial quantities, it is generally important to dissolve the starting material ;n a non-
polar solvent. Preferably, the starting material is presen~ in a concentration of 3M or less~
and more preferably, in a concentration of 0.01M to lM. Also. as stated above,
temperatures below 35~C are generally required. More preferably, the temperature used
with highly non-polar solvents, such as hexane, pentane, or chloroform, is in the ranee from -
25, 150C to +25~C. Still more preferably, the temperature used is in the range from -7~C
to -30 C.
The ozone may advantageously be generated with any of ~he commercially-availableozone generators. Such devices include corona discharge tubes through which oxygen gas
may be passed, For example, pure oxygen gas passing through an ozone generator will
typically leave the device as from 2% to 6% O3 (ozone), with the remainder 2 This ozone
mLxture may then be sparged through the terpene or non-terpene unsaturated hydrocarbon
starting materiai at a preferred temperature until the reaction is complete. Completion may
be judged by analyzing the gas exiting the ozonation chamber for ozone. (This may be done
by passing the exit gas through aqueous potassium iodide and determinine whether iodine
W(~ 93~126~4 PS:~US92/1û97~
-10-
61 S gas is liberated, or by any other conventional technique.) Alternatively, the reaction may
- be followed by observing the weight gain of lhe ma~erial undergoing t~ ~ reaction, by
observing changes in physical characteristi~s (such as conversion from a liquid forrn to a soft
paste), or by simply calculating the quanti~r of ozone needed to fully ozonate the material
S and stopp~ng the reaction when a slight excess of ozone has passed through the reaction
chamber.
When the starting material is normally a solid, such as ~carotene, it may be
solu~ilized in any sui~able saturated nonaqueous sohent system prior to ozonation. With
all of the diperoxide and trioxolane compounds, it is desirable to exclude water, lower
alcohols, nucleophilic peroxides, and proton donors from the reaction mLxture and from the
final composition, in order to prevent premature hydrolysis of the trioxolane or diperoxide
structure.
The following example shows a representative protocol for production of trioxolane
and diperoxide derivatives of unsaturated hydrocarbons.
1~ EXAMPLE I
P~otocol fnr Product}on of Trioxolane an~iperoxide D~rivativ~s
Ozone was prepared with an ozone generator. Dry oxygen con~aining about 10%
ozone was introduced at a speed of 10-20 liters/hour in an unsaturated hydrocarbon
solution. This solution consisted of the cis isomer of the hydrncarbon in dry and olefin free
butane, pentane or n-hexane as solvent. After ozonation, the solvent was removed at 30C
under rstation. The residue was either distilled in v~cuo or purified by column
chromatography on silica gel. The results for various unsaturated hydrocarbons of the
general structure RCH=CHR' are shown in Table 2.
TABLE 2
B R' Solvent Cone. (M~ T~(~ Yield ~%
t-butyl t-butyl pentane 0.3 -75 82
isopropyl isopropyl pentane 1.0 -70 85
ethyl ethyl pentane 0.2 -30 88
methyl methyl butane 0.2 -30 72
isopropyl methyl pentane 1.0 -70 86
ethyl methyl pentane 1.0 -70 91
propyl methyl pentane 1.0 -70 92
W093/12654 21~67S7 PCr/US92/~979
t-butyl methyl pentane 1.0 -70 81
t-butyl ethyl pentane 1.0 -70 84
ln addition to compounds of the structure RCH=CHR', we have also su~jected
S cycloolefins to ozonation using ~he general protocol of Example 1. Such cycloolefins can be
ozonated at a concen~ration of 3.0M in n-llexane at -7Q C to produce a yield ofapproximately 96%. However, the product of ozonation of cycloolefins tends to be peroxide
derivatives and/or insoluble polyrners of trioxolanes in inactive solvents, such as pentane.
Soluble trioxolane compounds can be formed ~rom ~ycloolefins using active solvents such
lû as ethyl acetate or acetone. The active solvent will enter into the resulting trioxolane
composition to produce a soluble monomer.
Acydic conjugated dienes and other polyunsaturated hydrocarbons can also be
ozonated to yield pharmaceutically active compounds for use within the scope of the present
invention. For example, acyclic conjuga~ed dienes can be dissolved in pentane a~ 0.8M at
a temperature of ~78C to produce a yield of approximately 74%.
Procedures other than ozonation are also kn~wn which can result in the
production of either the trioxolane or diper~xide derivatives. For example, non-ozonation
procedures for ~he production of methyl ethyl ketone diperoxide, diethyl ketone diperoxide,
1,1-dimethyl-4,4-diethyl-2,3,4,5-tetroxacydohexane and 1,4,4-trimethyl-1-ethyl-2.3,5,6-
te~raoxacyclohexane are described in Murray et al., supra.
EXAMPLE 2
Examination of Conditions for Production of Tri~xolane and Dip~roxide Derivatives
The ozonaeion protocol of DeVillez (U.S. Patent No. 4,451,480) and that of
Example 1 were each used on a sample of erucic acid methyl ester (a non-terpene
unsaturated hydrocarbon) and on a sample of jojoba oil (a terpene unsaturated
hydrocarbon). ln the DeVillez protocol, neat samples were ozonated at ambient
temperature (approximately 20~C). ln the Example 1 protocol, 3% samples in chloroform
were ozonated at -30C. Trioxolanes and diperoxides have a greater dipole moment than
the unozonated compounds which substantially increases their retardation factor (Rf) upon
chromatography. Thus, after ozonation, all four samples and a sample of each of the
unozonated compounds were chromatographed according to the method described bv
DeVi~lez using chloroform as a mobi~e phase and high performance silica gel as the
WO 93/126~4 PCI'/VS~2/1~97
2/~ ~6r~ 12-
- stationary phase~ The resulting plates were charred with iodine for identification. The
results are shown in ~able 3.
.
TABLE 3
Compound Rf
Unozonated Erucic Acid Me~hyl Ester 0.9
O~onated Erucic Acid Methyl Ester (DeVillez) 0.8, 0.9
Ozonated Erucic Acid Methyl Ester (Example }) 0.4, 0.5
Unozonated Jojoba Oil 0.6
Ozonated Jojoba Oil (DeVillez) 0.5, 0.6
Ozonated Jojoba Oil (Example 1) 0.I, 0.2
.
It can be seen from the results in Table 3 that the method of DeVillez results in the
forrnation of two spots upon chromatography, one of which appears to have the same Rf as
15 the unozonated rompound, and the other of which is only slightly re~arded. These n~o spots
are believed to represent the unreacted compound and a peroxide derivative ~hereof ~R-C-
O-O-CR'). In contrast, the method of Example 1 results in the formation of ~wo spo~s,
both of which are greatly retarded from the unozonated compound. These two compounds
are believed to represent the trioxolane and diperoxide derivatives of the compounds.
20 Pharmaceutical ~om~nsitions
ln one preferred embodiment of the present invention, the compounds of the
present invention are formulated into pharmaceutical preparations. These pharmaceutical
preparations include one or more of the trioxolane or diperoxide derivative compounds of
the presens invention, and may further include other pharmaceutically active ingredients.
25 In addition, any of the well-known pharmaceutically-acceptable carriers or excipients may
be combined with the compounds of the present invention in a well-known manner. Suitable
diluents include, for example, polyethylene glycol, isopropyl myristate, and mineral oil. The
pharmaceu~ical composition may be in any form suitable for topical use, such as an
ointment, gel, or cream. Conventional coloring, fragrance and preserving agents may also
30 be provided.
The excellent weight to oxygen ratio of some of the trioxolane or diperoxide
derivatives of unsaturated hydrocarbons renders them especially effective in treating many
medical conditions. TrioxoJane or diperoxide derivatives of highly unsa~urated hydrocarbons
are capable of releasing large amounts of oxygen, up to 30% of the weight of the compound
~O 93/12654 2 1 2 ~ 7 5 7 PC~/US92/10979
- -13-
and more. The trioxolane derivatives have three oxygen atoms at each site of unsaturation~
while the diperoxide derivatives have four oxygen iatoms. In addition, the trioxolane and
diperoxide derivatives used in the present invention appear to have significant unexpecled
- pharmacological properties that are different in kind or quality from those of unrelated
S ozonated compounds disclosed in the prior art.
The effective dosage of the compounds of the present invention appears ~o be
much lower than would be expected in light of the prior art, suggesting that the sompounds
have unexpectedly high efficacy. While the compounds may be used neat (and. indeed,
some of them form pharmaceutically elegant creams or oi~tments, e.g., linalyi trioxolane or
diperoxide derivative and linalool trioxolane or diperoxide derivat*e), the effective
concentration for most topical applications can be as little as 0.01%, by weight. However.
the compositions more preferably contain from about 0.5% or 1% to about I0% or 20% by
weight active ingredient. Topical compositions containing about 2% or 3~o of ac~ive
ingredient appear to be particularly effective.
For systemic use, such as intravenous, intramuscular, or intraperitoneal injection,
as well as rectal suppositories, the compositions may similarly contain from abvut 0.01% to
about 99% active ingredient, by weight. Preferred systemic compositions contain from about
0.05% to about 20% active ingredient, by weight.
The present invention further includes other sui~able pharmacological
preparations of trioxolane or diperoxide deriva~ives including: medicinal douches, eardrops,
eyedrops, throat sprays, sublingual preparations, dental preparations ~or topical sores.
mouthwashes, toothpaste, armpit deodorants, disinfectant/germicidals, germicidal soaps. and
contact lens steri~ization solutions. In addition, in certain embodiments of the invention. the
trioxolane or diperoxide derivatives are applied to a condom.
25, Other pharmaceutical preparations within the scope of the present invention
contain active ingredient in forrns, in addition to the foregoing, that are suitable for oral
phalyngeal use, such as oral rinses, trouches and medicated chewing gum.
In certain especially preferred embodiments, the pharmaceutical ~preparations
take a form which will provide for release of active compound in the intestine for intestinal
absorption. Examples of such intestinal-releasing forms include lozenges, capsules and gel
caps. ln any of these preparations, the active ingredient is preferably provided in a form
- appropriately coated to evade d;gestion by stomach enzymes to provide for intestinal
release. as is well known by those having ordinary skill in the pharmaceutical arts. However.
WO 93/12654 PCr/US92/10979- i
in certain embodiments, uncoated formulations, including oral elixirs, are believed tO release
- sufficient active ingredient to provide the desired pharmaceutical activity.
Thus, Example 3-9 are provided to illustrate certain pharmaceutical compositionswithin the scope of the presen~ invention. As such, ~hese examples are not intended to limit
the invention.
EXAMPLE 3
A va~inal suppositon ~or treasment of va~initis
2% w/v Ozonated linalyl acetate, from Example 2
Balance Hydrogenated vegetable oil base
EX~lPLE 4
A t~pical ~el efrective against burns
1% w/v Geraniol trioxolane
60% w~v Carbomer 934
1% w/v Disodium edetate
10% w/v Glycerin
Balance Polyethylene glycol m.w. 400
EXAMPLE 5
A toothpaste el~ective against gin~iYitis
1% w/v Trioxolane derivative of c~ 3-hexene-1-ol
Balance Conventional toothpaste formulation
EXAMPLE 6
A topical cream efrective a~ainst acne
2.5% w/v Linalool trioxolane
48% w/v Propylene glycol
25, 30% w/v Propyl paraben
5% w/v Polysorbate 60
10% w/v Glyceryl monostearate
Balance Mineral oil
EXAMPLE 7
A lubricant for condoms effecti~e a~ainst the transmission of STDs
0.~ g/ml C)zonated erucic acid
}C% w/v Glyceryl stearate
1% w/~ Food-starch modified
WO 9~/12654 2 I 2 6 7 5 7 PC~/US92/10979
~ .
2% w/v Polyethylene glycol m.w. 800
balance Light mineral oil
EXAMPLE B
An injectable composition effectiYe a~ainst Yerrucae
525 mg/ml ozonated linalyl acetate from Example 2
balance Polye~hylene glycol m.w. 200
E~AMPL~: 9
A r~al suppositon~ effective ~gainst sYstemic disorders
250 mg/ml Trioxolane derivative of CL5 3-hexene^l-ol
2 ml Pluracols (a rnixture of high molecular weight polyethylene glycol~ EXAMPLE 10
A rectal suppositor~ e~ti~e a~insl ~Ystemic disorders
250 mg Geraniol Trioxolane
1.5 ml Cocoa butter with bees' wax
15The toxicity of the trioxolane and diperoxide derivatiYes appears to be surprisingly
low, in both topical and systemic use. Our preliminary data suggess that the LD50 for a
representa~ive compound, linalool trioxclane is about 3000 mglkg in mice.
We have discovered that the trioxnlane and diperoxide derivatives of terpene andnsn-terpene unsaturated hydrocarbons of the present invention, when applied topically in
20suitable pharmacological compositions, are effective for treatment of bacterial, viral,
protozoal and fungal infections and for treatment of a variety of inflammatory conditions.
ln this regard. we have diseoYered that topical administration of the trioxolane or
diperoxide derivatives of the present invention, in a suitable composition having from about
0.1% to about 50% active ingredient by weight, preferably about 0.5% to about 20% by
25,weight, is effective to minimize the extent and severity sf Herpes simplex, genital herpes~
and chicken pox Jesions, when applied on incipient eruptions.
We have also discovered that vaginal administration of a composition con~aining the
trioxolane or diperoxide derivatives of the present invention, in a suitable vaginal carrier
(such as a suppository, cream, gel, or ~oam) having from about O.O5~c to about 90% active
30ingredient. by we;ght, preferab~ about 0.1~o ~o about 20% by weight, is substantially non-
irritating to mucous membrane tissues, and is effective to treat both bacterial and fungal
vaginal infections,
Furthermore. we have discoYered that topical administration of the trioxolane ordiperoxide deri~atives of the present imention, in a suitable composition having from about
WO 93/12654 PCr/VS92/10~7
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,6~ 0.01% to about 99% or 100% active ingredient, by weight, preferably from about 0.1% to
about 25% by weight, is effective in treating fungal infections of the skin and nails. such as
athlete's foot and onychomycosis. Similar compositions appear to have a shriiiking effect
on indo}ent neoplasms, including warts and moles.
S Compositions having from about 0.01% to about 50% active ingredient. preferab~y
about 0.1% to about 20%, are non-irritating to acne affected skin, and have exhibited a
strong anti-comedonal effect when used topically on affected areas. ~t is belieYed that these
compositions deliver nascent oxygen to kill anaerobic bacteria such as P. a~ne when the
trioxolane or diperoxide derivatives undergo hydrolysis. Furthermore, while it is not
intended that the applicants be limited to any particular theory or mode of action, it is
further believed that the particular ozonolysis fragments (such as ketones or carbo~yiic
acids) formed by tr~oxolane or diperoxide derivatives upon release Qf oxygen have a
complimentaly pharmacological effect.
Moreover, our data further indicate that topical application of the trioxolane or
diperoxide derivatives of the present invention, after significant exposure to the ultravio!et
component of sunlight, is effective in ameliorating the seYeri~y of sunburn and îacilitating
the healing process. Similar reduction of pain, inflammation, and blistering, and an increase
in the speed of the hea}ing process has been observed when the composition of the present
invention is applied to first and second degree thermal burns on a mammal.
Based on the den:onstrated antiviral, antifungal, and antibacterial properties of the
present compositions in vitro, and the relatively non-irritating properties of the trioxolane
- or diperoxide derivatives, it is further believed that topical administration of ihe compounds
of the present invention can decrease the probability of transmission of sexually transmitted
diseases (STD's). Thus, for exampJe, the previously described vaginal compositions may be
25~ used alone or in conjunction with a condom to decrease the risk of in~ection. In this regard.
the active ingredient may further advantageously be formulaled into a lubricating
composition of known type.
Furthermore, we have discovered that the trioxolane and diperoxide derivatives of
the present invention are effective spermicides. Thus, intravaginal application thereof can
3D serve to minimize the chances of pregnancy as weJI as to prevent the transmission of STD's.
We have also discovered that topical administration of trioxolane or diperoxide
derivatives in a topical preparation exhibits significant efficacy in the treatment of most
dermatoses, including psoria~Js and those dermatoses caused by bee stings, insect bi~es.
poison plants such as poison oak, poison i~y, and stinging nettle, diaper rash. hives, and
WO 93/12654 2 1 2 ~ 7 5 7 PCT/US92/1~979
other reactions for which antihistamine or steroidal medications are commonly prescribed.
Admini~ration of the trioxolane or diperoxide derivatives of the presen~ invention in lieu
of steroidal medications is sometimes equally effective; however. side effects are
considerably reduced, making therapy with trioxolane or diperoxide derivatives the more
desirable treatment. The invention, however, con~emplates combination therapy in some
instances. Thus, in addition to an effective amount of trioxolane or diperoxide derivative,
the compositions of the present invention may further indude an effective amount of an
antihistamine or a corticosteroid. These medications are well known, and ef~ective dosages
for the various antihistamines and corticosteroids have been established. When used
together with a trioxoJane or diperoxide deriYative, the effective t~pical concentrations of
these ingredients will generally be toward the lower end of the effective range in which they
are presently used alone.
The present invention also includes methods of systemic and localized injection of
the compositions disclosed herein, including intravascular. intramuscular, subcutaneous.
intraperitoneal, and other injec~ion techniques. Such injecaion may be used for treatment
of viral, ~ungal, and bacterial infection. We have also discovered that locali~ed injection of
a trioxolane or diperoxide derivative cf the present invention into a tumor has an anti-
neoplastic effect.
Other embodiments of the present invention provide methods of oral pharyngeal
treatment for sore throat and/or oral infection caused by, for example, viral, bacterial
and/or fungal infection, including candidiasis. In these methods. the active ingredient is
applied locally to the affected oral pharyngeal area, ordinarily through ayplication of an
appropriate pharmaceutical preparation containing active ingredient, such as a mouthwash.
throat spray, oral rinse, troche or medicated chewing gum.
25~ A particularly preferred method of treatment for infection of the gastro-intestinal
tract, as well as for treatment of systemic infection in general, is through application of a
pharmaceutical preparation which will provide for release of active compound in the
intestine for intestinal absorption. As discussed above, these preparations are preferably
appropriately coated to evade digestion by stomach enzymes to provide for intestinal
release. For systemic treatment through intestinal absorption, the amount of active
ingredient is preferably administered at from 0.2 mg/kg body weight to 400 mg/kg body
weight per day, more preferably from 2 mg/kg body weigh to 200 mg/kg body weight. The
daily dose can be given in a single dose or divided into two or more equal or unequal doses.
WO 93/12654 P~r/US~2/1097
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6~1~ EX~MPLE 11
Test for EFrlcac~ of T~eatment of Sunburn
The composition of Example~ 4 is applied topic~lly to onJy a portion of the skinsurface of a severely sunburned patient in a single application, ~wo hours after the exposure
to sunlight. The treated area exhibits slight reddening, but no peeling or blistering. Only
minor discomfort is apparent. The un~reated area, in contrast, becomes red, blistered, and
painful.
} :XAMPLE 12
l~s~ for EmcacY o~ Treatment o~ ~icken P~x
The composition of Example 6 is tQP;~1IY applied to a portion of the lesions on a
child suffering from chicken pox. Within 1 hour, the sreated lesions are significantlv reduced
with little or no self-induced trauma ~rom scratching. The untreated lesions are unchanged
in size, and show the effects of trauma frorn scratching.
EXAMPLE 13
Test for EfficacY o~ Treatment of Swollen loints
Patients a~ a spor~s medicine clinic complaining of s~ollen knees are divided into
three groups: groups A, B and C. The patients in group A receive an injection of the
composition of Example 8 into the swollen knee. The patients in group B receive an
injection of a placebo, the composition without active ingredient. The patients in group C
receive an injection of a corticosteroidal medication. Within 12 hours the swelling in the
knees of the patients in group A is significantly reduced. No change is reported in the
knees of the patients of group B. The swelling in the knees of the patients of group C is
also reduced, however, a significant percentage of the patients suffer inflammatorv reactions.
EXAMPLE 14
2~, Test for E5~;cacY o~ Treatment of Funeal ln~ections Or ~he Va~ina
The suppository of Exampie 3 is administered intra-vaginally to one group of
patients suffering from yeast infections of the vagina. A second group of such patients
receive a suppository without the active ingredient of Example 3. A third group receives
a suppository containing the drug clotrimazole, a commonly used drug for treatmen~ of
3~ fungal infections of the vagina. Every 24 hours the process is repeated. Within 2 days, the
patients of the first group have no reddening of the vagina and within 7 days, a yeast culture
produces negative results. The second group of patien;s continues to complain of itching
and other common complaints of fungal infections. A yeast assay is positive. For pat;ents
WO 93/12654 21 ~ 6 l 5 7 P~/VS92/10979
.
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in the third group, a yeast assay is negative; however, a number of these patients complain
of irritation and in those patients, a significant reddening of the vagina is present.
lEXAMPLE 15
In vitro anti-microbial assa~ of lina1001 trioxolane
S A culture of E. coli was har~ested with sterile saline using sterile swabs. The
nurnber of Colony Forming Units (CFUs) per ml in the suspension was deterrnined by
Standard Plate Count Method. A working suspension of E. coli with approximately 1.0 X
107 CFlJs/0.1 ml was then prepared. Four aliquots of 1 ml each of test oin~ment containing
1.0% trioxolane or diperoxide derivative of linalool were removed and placed in separate
sterile screw-capped tubes. Each sample was inoculated with D.1 ml of the working
suspension of E. coli to yield a final concentration of approximately 1 X 10S CFUs/1 ml of
the product. The samples were stored at 20 - 2~C for a total of 28 days. Samples were
selected at 7 day intervals to determine the number of viable CFUs present. A control with
uninoculated ointrnent was also stored with samples selected at the same intervals. A~ 7
days, and aJl subsequent sample selections, there were less than 10 CFlJs present. No CFUs
were present in any control sample.
EXAMPLE 16
Priman skin irri~ation test of trioxolane or diperoxide deriYative of linalool
Six healthy New Zealand yrhite rabbits were tested ~or skin irri~ation.
Approx;mately four hours prior tO application of the trioxolane or diperoxide derivative
sample, the backs of the animals were clipped free of fur. Each rabbit received epidermal
abrasions with a sterile needle at one test site while the skin at another ~est site remained
intact. A 1.0% solution of linalool trioxolane or diperoxide derivative in isopropyl myristate
was prepared. A 0.5 ml portion of the test solution was applied ~o each si~e by introduction
25, under a double gauze layer to an area of skin approximately 1" square. The patches were
covered with a nonreactive tape and the entire test site was wrapped with a binder. After
24 hours. the binders. tape, and test material were removed and the skin evaluated. The
test material residue was removed with 70% isopropyl alcohol. An evaluation was also
made at 72 hours after application. The reactions were scored according to the methods
described in the Federal Hazardous Substances Act. The test solution had a Primarv
lrritation Index ~PII) of 1.~. According to F~ISA regulations, a material with a PI1 of less
WO 93~12654 ` PCr~ i92/1097
-20-
~,~?,6~ than 5.00 is ~enerally not considered a primary irritant to the skin.
EXAMPLE 17
C)cular ;ITitation test in the rabbit oî linalool trioxo!ane
Six healthy New Zealand White rabbits were selec~ed for study. The rabbits' eyeswere judged free of irritation prior to the study by examining with a pen light and under UV
light after installation of 2% fluorescein s~ain. A 1% solution of ~he trioxolane or
diperoxide derivative of linalool was prepared in isopropyl myristate. A 0.1 ml portion of
this test solution was instilled into the lower conjunctival sac of one eye of each rabbit. l he
lidswere held closed for one second. The opposite eye of each rabbit received 0.1 ml of the
isopropyl myristate, as control. Eyes were examined and the ocular reaction scored
according to the "lllustrated Guide for Grading Eye Irritation by Hazardous Substances"
~Appendix 1). At 24, 48, and 72 hours post docin& the eyes were examined with a pen light
and re-examined with UV light following fluorescein staining of the cornea. Under ~he
conditions of this test, the test solution was considered a non-irritant to ocular tissues of the
rabbit.
Saudies on Deri~ratives of a Terpene and Non~Terpene lInsatura~$d Hvdroc~rbon
1. TNTRODl~CllON
Geraniol trioxolane, a trioxolane derivative of a terpene, was assessed on the basis
of i~s direct biological activity against certain target organisms in vitro. In vivo assays were
2û similarly undertaken to assess the product's ~oxicity, safety, and in certain cases~ efficacy.
The studies were conducted using product manufactured using the synthesis protosol of
Example I, and synthesized strictly in accordance with international guidelines and
specifications, especially those issued by Untied States Food and Drug Administration.
United States Pharmacopoeia, British Pharrnacopoeia, Kenya Association of Manufacturers,
and Pharmacy and Poisons Board of Kenya. llle preparatory process met all requirements
for the Code of Good Manufacturing Practices (GMP). In addition. the trioxolane
derivative of cis 3-hexene~l-ol, a non-terpene, was s~nthesized and compared to Geraniol
trioxolane ior the purposes of assessing its comparative activity.
Both Cieraniol trioxolane and the trioxolane derivative of cis 3-hexene-1-ol arecolorless liguids. Geraniol trioxolane is more viscous and less stable than ~he trioxolane
derivative of cLs 3-hexene-l-ol.
WO 93/126~4 2 1 2 & 7 5 7 PCI/VS~2/10979
.
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2. ~N VI7RO EXPERIMENTAL STUDIES
2.1 Effect of Geraniol trioxolane and the trioxolane derivative of cis 3-hexene-1-ol on
Leishmania donovani and Leishm~nia major promas~otes in vitro
- 2.1.1 Geraniol tr;oxolane
S This was first diluted in PEG600 to give 250 mg/ml, and further diluted in culture
rnedium to give a working concentration of 62.5 mg/ml. lllrough serial dilu~ions~ the
compound showed killing of all promastigotes up to a dilution 1:2048 within 18 hours. The
controls in the same titre plate survived.
2.1.2 Trioxolane derivative of as 3-hexene-1-ol
This compound was ~lrst diluted to 400 mg/ml in PEG600, and further serially
diluted in culture medium. The compound caused the killing of all promastigotes within 18
hours up to a dilution of 1:28-29.
2.2 Effect of Geraniol trioxolane and the trioxolane derivative of CLS 3-hexene-l-ol on
myeloma cell line and spermatozoa
The susceptibili~y of Myeloma cell line (x63 balb/c line) and human spermatozoa
to both Geraniol trioxolane and the trioxolane derivative of cis 3-hexene-1-ol was assessed.
The myeloma cell line was killed within 48 hours and human spermatozoa were killed w;thin
1 minute, at dilutions of less than 1:2x101 of the working concentrations.
2.3 Effect of Geraniol trioxolane and the trioxolane derivative of cis 3-hexene-l-ol on
microor~anisms
It was decided to test the direct biological activity of both Geraniol trioxolane and
the trioxolane derivative of cis 3-hexene-1-ol on microorganisms which cause common
infections in our community. Thus, the foDowing microorganisms were tested for their
susceptibility to these compounds:
2.3.1 Diarrhoeal diseases
i) Salmonella spp.
iu) Shigella spp.
iui) Enteropathogenic/enterotoxigenic Escherichia coli
2.3.2 Urinarv tract infections both bacteria and fun~i
~0 i) Neisseri~ gonorrhoea (PPNG 3nd ns~n-PPNG)
ii) Candida albicans
iu) Pseudomonas spp.
WO 93/12654 PCI~/U~7"~-,
6~ 22- " 2.3.3 Bacteria causin~ respiratorv tract infections
i) ~lebsiella spp.
ii) Sf~p)ylococcus aureus
iii) Staphylococcus epiderrnidis
23.4 Other infections caused by bacteria
i) Pr~teus
ii) Achr~mobacter
iii) E. coli
2.3.5 Fun~al inf,e,ction
i) Commc~n dematophytes
a) 7richophyton violaceum
b) Tnchophy~on canis
u) Systemic fungi
a) C~yp~occu3 spp.
b) Can~ spp.
iii) Other general fungi
a) PhialopJ~ora venucose
b) Penicill~um spp.
2.4 Results
Minimum inhibition concentration (MlC), as well as minimum bacterial
concentration (MBC), of the drugs on the common pathogens were determined. Standard
drugs, and in certain cases reference stains, were used as controls and for comparative
purposes. Results of observations are summarized in Tables 4-6.
2~267~7
WO 93/12654 ~ PCI /lJS92/10979
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TABLE 4
MIC of Geraniol trioxolane and the trioxolane derivative of cis 3-hexene-l-ol on ~arious
common pathogens by agar diffusion method
s
¦ Organism N MlC (I ng/ml)
l Geraniol trioxolane Trioxolane derivative
I of cis 3-hexene-1-o~
I - .,. ~ ... . . ._ ,
¦ Gram positive cocci
I S. aureus 16 0.31 (0.004)~ a25 (0.031)
I S. epide~mi~s 14 0.31 (0.004) 0.025 (a.031)
I . _ .
Gram ne~ative cocci
M g~norrhoea 49 0.15 (0.00~) 0.003~ (0.00I9)
. . _ .
Gram ne~.ative bacilli
l Salmonella 3 031 0.007
¦ Shigella 7 0.31 0.25 (0.031)
EPEC/ElE~: 16 052 (0.31) 0.25 (0.031)
PseudomonQs 2 0.035 0.007
P~ebisella 1 0.017 0.007
E. coli 3 0.31 0.12
Archromobacter 1 0.62 0.06
. , , ,, . _ . ..
un~,i
C albicans 5 0.62 0.031
Tn. violacel~m 1 0.31 0.15
7ri. canis l 0.31 0.15
C~yptococc~s 1 0.15 0.015
Ph verrucose 1 031 0.15
Penicillium 1 0.62 0.31
~ _ ~
Note: * numbers in parentheses indicate the lowest concentration inhibising at least one
isolate of organisms
N = number of isolates tested
MlC = minimum inhibition concentration
EPEC/ETEC = enteropathogenic Escheridlia coli/enterotoxigenic ~sc~lerichia coli
WO 93/12654 PCr~US92/~097~
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?. TABLE 5
MIC of Geraniol trioxolane and the trioxolane derivative of cis 3-hexene-1-ol on some
common pathogens in agar dilution
,; , . .~ __ _
Organism N MIC (rng/ml)
Geraniol Trioxolane
trioxolane derivative of
3-hexene-1-ol
. agar broth agar broth
S. aureus 100.15 0.78 0.12 0.62
EPEC/ETEC 50.62 0.56 0.031 0.62
~ . ........... . . . .
Salmonelfa 3 0.31 3.12 Q.0û7 0.15
Shigella 7 0.31 1 .56 0.007 0.62
Pseudomona3 2 0.035 1.56 0.007 0.62
~ 2 0.62 3.12 0.031 ~ 0.31
Note: ~ numbers in parentheses indicate the lowest concentration inhibiting at least one
isolate of organisms
N = number of isolates ~ested
MIC = minimum inhibition concentration
EPEC/ETEC = enteropathogenic Esc~teric~ia coli/enterotoxigenic Esc,teridtia cofi
WO 93/12654 2 1 ~ 6 7 5 7 P~/IJ!~;92/10979
: ?
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TABLE 6
MBC of Geraniol trioxolane and the trioxolane der;vative of cis 3-hexene-1-ol on various
Scommon pathogens by broth method
- . ..... _ ~ I
Organism N MBC (mg/ml~
. . . . ; .. ..
C;eraniol trioxolane Trioxolane derivative
of cis 3-hexene-1-oJ
. . . _ . . ~ .
Gram positive cocci
10S. aureus 10 3.12 (0.78)~ 125
5. epiderrnidis
, .......... ... . . . ~.
Gram ne~ative bacilli
Salmonella 3 6.25 2.5
Shigella 7 3.12 1.25
EPEC/ElEC 5 3.12 1.25
Pseudomonas 2 3.12 1.25
Fungi _ .
Can~da 2 3.12 125
~ .. . . ~
Note: ~ numbers in parentheses indicate the lowest bacterial concentration for
corresponding organism
N = number of iso~ates tested
l~qBC = minimum bacterial concentration
EPEC/Ell~C = enteropathogenic Escherichia coli/enterotoxigenic Eschenchia coli
~t can be seen from the results summarized in Tables 4-6 that both Geraniol
trioxolane and the trioxolane derivat;ve of cis 3-hexene-1-ol are more ac~ive than
conventional drugs against commonly encountered microorganisms (in vi~r~). Thus. the
derivatives were ~ound effective against the following microorganisms which cause common
infections:
a) diarrltoea (Salntonella 5pp., Shigella spp., enteropathogenic/enterotoxigenic Eschenc~tia coli)
b) urinary tract infections (Neisseria gonorrJtoea, candida spp. )
c) respiratory tract infections (~lebsiella spp., Staphylococcu~ spp. )
d) fungal infections (7richophyt~n spp.. CrytOCOCCtl5 Spp., P~tiLtlophora Spp.,
Penicil/ium 5pp.).
The diluent, propylene glycol, did not inhibit the growth of either bacteria or fungi.
Water significantly reduces the direct bioactivity of hoth Geran;ol trioxolane and the
WO 93/1~6~4 PCI/US92/1097P
-26-
6~ 1 trioxolane derivative of cis 3-hexene-1-ol. In comparison, 100% propylene glycol is a better
~,~1 diluent ~han water.
The trioxolane derivative of CLS 3-hexene-1-ol appears to be more active than
Geraniol trioxoiane~ and the direct bioactivity of these drugs wer~ better in agar than in
S broth methods. However, the actiYity of both compounds on microorganisms has no
relationship with resistance and sensitivity of conventional antibiotics on bacteria. All the
microorganisms tested (including both gram positive and gram negative bacteria) were
uniformly sensitive io the two prodllcts.
3. IN V~VO EXPERIMENTAL STUDIES
lû 3.1 Tolerabilitv of mice tO Geranio} trioxolane and the trioxolane derivative of cis
3-hexene-1-ol
20g balb/c mice received various concentrations of both Geraniol trioxolane and the
trioxolane derivative of Ci3 3-hexene-1-ol intraperitoneally (IP). The minimum lethal dose
was observed at 3g/kg. Studies on LD50 revealed ~hat the products are highly tolerated.
3.2 Immunomodulatorv Activitv of Geraniol trioxolane and the trioxolane derivative of
cis 3-hexene-1-ol
A group of 15 Balb/c mice which had been used as controls in the previous
experiments on Leishmania infection were studied further to assess the astivity of bosh
Geraniol trioxolane and the trioxolane derivative of cis 3-hexene-1-ol in the immunological
status of these mice.
Bone marrow examination revealed that ~here was an increase in new clones of
lyrnphocytes in mice put on either Geraniol trioxolane or the trioxolane derivative of cis
3-hexene- 1 -ol as compared to mice without any drug. This observation strongly suggests that
both Geraniol trioxolane and the trioxolane derivative of CLS 3-hexene- 1 -ol are
25, immunomodulators, a property which is of critical consideration in the treatrnent of diseases
that are associated with immunosuppression.
3.3 Treatment of mice infected with Leishmania maior
Efficacy of Geraniol trioxolane and the trioxolane derivalive of cis 3-hexene-1-ol in
the treatment of cutaneous leishmaniasis was assessed in balb/c mice experimentally
infected with Leishmania major. The compounds were used intraperitoneal~y (IP) and
topically, and further sompared to the standard anti-leishmanial regimen ~Pentostam. an
antimony-based formulation) and controls.
~O 93/12654 21 2 b 75 7 PCI /US92/10979
The topical preparation was forrnulated as an ointment containing 4 mg/ml of
compound and using û.I ml per lesion per mouse, whereas the JP preparation contained 0.4
mg in 0.5 ml of compound per mouse. There were a total of 15 mice per treatment group.
The summary of obser~ations is given in Table 7. It is clear that miee treated with
5the topical preparation responded better than those in any of the other treatment groups.
T~BLE ?
Efficacy of Geranio~ trioxolane, the trioxolane derivative of cis 3-hexene-1-ol and
Pentostam in experimentally infected mice with Le~s*mania major
' . ... ,
Treatment Post-Treatment Observations
,
Group ` Day 0 Day 14
n= I5/group
,, . .
Controls Large lesions Lesions breaking
(infected) Palate l t
, _ - _ .
Pentostam Large lesions Large lesions breaking
(IP) Palate imolvement
~ . . _ . _ .
Geraniol trioxolane Large lesions Lesions ~reaking
(IP) 4 dead
~ . . . _ Il
ZO Geraniol trioxolane Large ~esions Dry lesions
(topica1) No palate involvement
No death
~ealthy
Trioxolane derivative of cis Large lesions Lesions scabbed
3-hexene-1-ol ~ palate involvement
(IP) 2 dead
.. _ . . . . ll
~ Trioxo~ane derivative of cis Large lesions Lesions small and dry
3-hexene-I-ol Animals healthy and active ¦
(topicaJ) No death
. = .,
3.4 Trea~ment of mice infected with Le hmania don~va
~alb/c mice were experimentalJy infected with Leishmania donova. the causative
agent of visceral ieishmaniasis. The infection was viscerali~ed in 3-4 weeks. lnfected mice
were divided into four groups of 15 mice each. One group was treated with Geraniol
trioxolane. another with the trioxolane derivative of cis 3-hexene-1-ol and the ~hird group
w;th Pentostam. The Jourth group was kept as a control group. i~ach medication was used
WO 93J126S4 PCr/US92/109
2~-
6 at 20 rng/kg body weight and given in 0.5 ml intraperitoneal~ daily doses for 5 days. The
average weights of mice were 20 g each.
Mice were examined after the S days of treatment and every week thereafter.
Indicators of response to treatment were examination of visceral organs îor the parasites
S in autopsied mice, plus the general well-being of Ihe living.
Preliminary results of this study indicate that both Geraniol trioxolane and thetrioxolane deri~ative of cis 3-hexene-1-ol perform be~ter than Pentostam in clearing parasites
from the vis~era, such as the spleen.
4. CLIN~CAL STUDIES IN ~UMANS
I0 4.1` ~linical Studies on Candidiasis
We have begun clinical studies to assess the efficacy of Geraniol trioxolane and ~he
triox~ane derivative of cis 3-hexene-1-ol in the treatment of common infections, including
those that are considered as common opportunistic infections in HJV-infected persons. The
patients recruited in the present studies are those who volunteered themselves to participate
in the studies.
Candidiasis is a particular opportunistic infection which is common in H~V infec~ed
individuals. Vaginal candidiasis is a very common infection suffered by virtually all women
at one time or another. Thus, we have perfonned clinical studies on this fungaJ infection
as foli~ws:
ZO 4.1.1 25 ml of a liquid preparation containing 25 mg/ml of the product was applied
three times a day in the oropharyngeal area in ten patients with oral candidiasis. The
infection cleared within 5 days and did not require add;tional ~reatment.
4.1.2 Vaginal inserts were formulated and used twice a day in five female
volunteers suffering from vaginal candidiasis, and a further three having both vaginal
2~ candidiasis and gram positive cocci infections. As in oral candidiasis, these infections
- cleared within 5 days and required no further treatment.
4.2 Clinical Studies on Svstemic Infections
4.2.1 AIDS: A minimum of 10 patients have been followed over a period of from
2 to 4 years on a dosage regimen of approximately 200 milligrams daily. All patients have
shown improvement and stabilization of both cJinical and Jaboratory parame~ers of disease.
4.2.2 CANCER: 4 patients have been treated oYer a period of from 2 to 4 years.
These incJude one Adenocarcinoma of the Lung, one }~odgkins Disease, and two
Lymphomas. Patients were treated on a daily dose basis of 20n milligrams daily. In all
patients, during the course of treatment, there was regression of tumor or cessa~ion of
.. . . . . .
3/126~4 2 1 2 6 7 ~ ~ P~/US~2/lOg79
.. . .
-29-
growth of tumor, as well as improvement in clinical parameters. One patient demonstrated
reinstitution of tumor growth with forced cessation of therapy. The other "atients remain
in remission.
4.2.3 RHEUMATC)IDARTHRITIS: 4 patientswith long-standing active RA were
S placed on a dose of 200 milligrams daily. All patients had evidence of remission of
symptoms within 2 weeks, and in all patients all evidence of aclive disease had cleared
within 6-8 weeks. Patients have remained asymptomatic with no evidence of progression
of disease for n~o years.
4.2.4 OSlEOARTHRITlS AND ~NFLAMMATORY POLYARTHRITIS: 10
patients, including those with degenerative, psoriatic and arthritis associated with chronic
fasigue synàrols~e, as well as non-specific polyarthritis were studied. These patients were
treated with a daily dose of 200 milligrams. All patients showed substantial or complete
disappearance of all syrnptoms within 8 weeks. All patients have been maintained Y,rithout
symptom and without evidence of progression of disease.
4.2.5 CHRQNIC FATIGUE SYNDROME: 4 patients were treated with 200
milligrams daily. All patients showed marked improvement. All symptoms were completely
or substantially cleared within 3 months.
4.2.6 LUPIJS ERYTHEMATOSUS: 1 patient wi~h associated colitis and arthritis.
All signs and syrnptoms of active disease cleared within 90 days with complete resolution
of arthritis, colitis and reduction of ANA titer from 1:1880 down to 1:30.
4.3 Clinical Studies nn Topical Diseases
4.3.1 FIJNGA~ D~SEASES OF THE SKIN: 30 patients were treated for various
forrns of slcin fungus unresponsive to other forms of therapy. All patients had complete
clearing of slcin lesions within 4-6 weeks. The lesions were treated with a 3% solution of
25, active ing~edient in propylene glycol once or twice daiJy.
4.3.2 BACIERIAL D~SEASES OF THE SK~N:
i) ACNE: ln a series of 40 patients with chronic acne vulgaris of va~ing
degrees of severity, a 3% solution was applied to the lesions on a daily basis. All patients
shows significant or marked clearing of lesions. New lesions formed less often and cleared
quickly with subsequent applications.
ii) lMPETlGO: 3 paticnts had complete clearing within 1 week with application
of 3% solution twice daiJy.
iii) PARONYCHlA: 4 patients. Lesions cleared rapidly and completelv in all
cases within 48 hours of beginning applicalion of 3~o solution.
WO 93/!~s4 PCr/US~2/l09~'`
~ -30-
- iv) WOUND HEAL~NG and SCAR PREVENTION: Various surgical and non-
surgical wounds were trealed with a 3% solution on a daily basis. Wounds so treated w~, ~
shown to heal faster, with no evidence o secondary infectinn and noticeable reduction in
scar formation. Those persons prone to keloid formation had no evidence of keloid
S forma~ion with the use of this treatment.
4.3.3 RAL DISEASES OFTHE SK~N:
i) HERPES: 40 patients comprising herpes simp~ex, genitalis~ zoster,
oph~halmic were treated with a topical solution of 3% active in~edient in propylene glycol,
and in the- case of ophthalmic with a fresh l/2% solution in saline wi~h a small amount of
propylene glycol as solubi~izing agent. In all herpes genitalis and simplex cases. all lesions
when treated ear~y showed rapid cessation of ~riral expression and rapid clearing of lesions
in most cases, without evidence of blister formation. Most lesinns cleared completely within
48 hours. Lesions which had progressed to significant size prior to treatment required 3~
days for complete resolution. Herpes zoster (5 cases), all cases showed slow progressive
resolution of lesions, with complete clearing in approximately 2-6 weeks. Most lesions had
been present for up over 6 months. Ophthalmic herpes (2 cases), in both cases lesions
showed definite early clearing, with resolution in one case within 48 hours and the other
case in 4 days.
ii) VENEREAL WARTS: 4 patients. A solution of 3% acti~e ingredient in was
applied 3 times daily with complete clearing of all lesions in 2~3 weeks.
iii) COMMO~ WARTS (MUI.nPLE MAN~FES~AllONS): 26 patients. 10%
solution was applied 2 times dai~y. All patients shows resolution with treatment: however.
there was marked variation in time to complete clearing, depending apparen~ly upon the
type of presentation of the wart. Some warts cleared within 2 weeks, while most required
several months and a few manifestations took over I year for complete clearing.
4.3.4 DERMATOLOGTCAL:
i) ECZEMA: 14patients. Variousformsofec~emaorneurndermatitiswere
treated and approximately 1/2 showed comple~e resolution of lesions within 6 weeks.
Approximately 30% showed significant improvement and the remainder showed only slight
or no improvement.
ii) PSORIASIS: 13 patients. A group of palients with various degrees of
severity were treated. Milder cases were treated with topical application of 3% solution,
and there was marked or complete resolution of lesions in all but 2 cases. 3 cases of severe
generalized derrnatitis were treated with a combination of 3% topical once or twice daily.
,, .
~WO 93/12giS4 2 1 2 ~ 7~ 7 PCr/US92/10979
or with systernic dosage of 200 milligrams 3 times weekly. All showed complete clearing of
lesions within 6-8 weeks.
iii) INSECT BlTES: Multiple cases of insect bi~es. including those of rnosquito.bee, wasp, fly, flea, totalling in excess of 20 patients were treated. All lesions showed almost
S immediate relief of pain, itching, with rapid reduction of swelling and erythema. All lesions
cleared completely within 24 to 4B hours.
iv) CC:~RAL BURNS/JELL~FISH STINGS: 18 patients. All pain, swelling and
erytherna was alleviated rapidly with either complete clearing or minor residual erythema
within 24 hours.
v) POISON OAK: 3 patients. Application of 3% solution provided marked
and immediate relief of itching, and in 2 cases with complete clearing of lesions within 4
hours.
vi) SEBORRHEIC DERMAl~TIS: 10 patients. Lesions involving the scalp and
eyebrows were treated with once daily applications with marked response and reduction of
lesions in 9 out of the 10 cases.
vii) BURNS: 19 patients with first and second degree burns. Most patients
reported immediate and substantial or complete relief of pain. Most patients showed
evidence of ~he deYelopment of buliae and there was rapid and substantial reduction in
swelling and e~hema in all patients. No third degree burns were treated.
4.4 Clinical Studies on Arthritis
4.4.1 ARTHRmS: S patients of relatively severe arthritis of small joints were
treated with 6% topical applications twice daily. All patients reported marked or complete
relief of pain within 2 weeks.
4.5 Clinical Studies on Dental and Oral Conditions:
4.5.1 PHARYNGlTIS: 17 patients with varying degrees of tonsillitis or pharyngitis
were treated with topical application of 3% active ingredient in glycerin 4 times each day.
All patients showed moderate to rapid relief of pain with reduction of swelling in the first
24 hours. Complete resolution was seen in all patients within 48 to 72 hours.
4.5.2 TOOTH ACHE: 7 patients with varying degrees of den~al pain from tooth
- 30 ache experienced immediate and substantial relief from pain with application of 3% topieal
solution to the surrounding gums.
4.5.3 G~NG~VlllS: 3 patientswith gingivitis had a marked or complete resolution
within 3 months upon using 3-4 drops 3% geraniol trioxolane in glycerine wilh toothpaste.
WO 93/12~54 PCI/US92/109~'`
-3~-
4.6 Chnlcal Studles on Oral Manlfestat!ons of Bacterla! Over~h:
4.6.1 HALlTOSIS: 10 pa~ients with chronic halitosis all reported marked or
complete resolution of the halitosis with daily use of four drops of 3% solution in glycerin
during brushing of teeth.
4.6.2 CANKER SORES: 9 patients. All patients reported rapid resolution of pain
and discomfort with complete clearing of lesions within 24 to 48 hours by application of 3%
solution 3 times a day.
4.7 GYNECOLOGTCAL:
4.7.1 SPERMICIDE: A pilot study using 4 couples using a vaginal suppository of
1.5% solution in high molecular weight polyethylene glycol (Plurocols) over a 4-month
program and there has been no evidence of conception to date.
4.7.2 HEMORRHOIDS: 21 patients with hemorrhoids ranging from small
external symptomatic hemorrhoids to large external and internal bleeding hemorrhoids. A31
cases showed substantial or complete resolution within 1 week using 3% topical solution or
1~ a combination of topical and rectal suppository.
5. CONCLVSIO~
From the data obtained so far, it is dear that the two products are effective against
an extremely wide spectrum of bacteria, fungi, viruses and protozsa. They have also been
observed to be sperrnicidal, as well as immunomodulatory. There is no reeognized toxicity
or undesirable side ef~ect associated with their use. These products are therefore believed
- to be potent drugs for use in a very wide range of activi~ies.
Both Geraniol trioxolane and the trioxoJane derivative of cis 3-hexene-1-ol are
potentially active compounds against a variety of organisms, notable ones being protozoa
~Leishmania spp.), both gram positive and gram negative bacteria, and fungi. An additional
activity observed in vitro is their ability to kill spermatozoa and cultured myeloma cells. On
the basis of the results from ongoing studies on safety and toxicity, the products are believed
safe for human use as drugs.
In addition to the clinical data obtained on Geraniol trioxolane and the trioxolane
derivative of CL~ 3-hexene-1-ol, we have obtained clinical data using erucic acid ozonated in
~0 accordance with the method of Example 1. This ozonated compound is active in Yitro
against a wide variety of bacterial and fungal pathogens. The compound has successfully
been used topically in a propylene glycol base at 2~o active material to treat genital and oral
herpes, fungal infecti~ns, wasp stings, eczema, and diaper rash. ln additiorl, a ~hree-month
trialon three ARC (AJDS Related Complex) patients and one Iymphoma patient. remission
W~93/126~4 21267~ 7 PCT/US9~10979
-33-
of disease was maintained after an initial treatment with geraniol trioxolane resulted in
remission.
The trioxolane and d;peroxide derivatives of 'ooth terpene and non-telpene
unsaturated hydrocarbons are all believed to have the same mechanism of action. While
not wishing tO be bound by any particular mode of action, it is believed that these
compounds release pharmacologically active zwitterion moieties upon hydrolysis. In view
of the compounds' perceived common mechanism of action, it is believed that a wide variety
of diperoxide and trioxolane derivatives of terpene and non-terpene unsaturated
hydrocarbons have activit;es similar to the activities reporsed in this section for Geraniol
trioxolane and the trioxolane deriYative of CLS 3-hexene-1-ol.
