OxyFile #173


United States Patent 4983637
Filing Period: Jan. 1, 1987 - Present
Application Number: 363628
Application Type: Invention (Utility) Patent
Application Filing Date: June 8, 1989
Title of Invention: Method for treating viral infection of HIV

Issue Date: January 8, 1991
Primary Examiner: Nutter; Nathan M.

Inventor: Herman; Stephen(9341 Hazel Cir., Villa Park, CA  92667).

References:

U.S. Patent Documents:

925590 (June, 1909; Neel)
1210949 (January, 1917; Knox)
1910564 (May, 1933; Rankin)
2083572 (June, 1937; McKee)
2243053 (May, 1941; Ramage)
2356062 (August, 1944; Johnson)
2750411 (June, 1956; Fisher et al.)
3360472 (December, 1967; Renold)
3504038 (March, 1970; Beal)
4451480 (May, 1984; De Villez)
4591602 (May, 1986; De Villez)
4632980 (December, 1986; Zee et al.)

Other References:

Russell-Manning, editor, "Self-Treatment for AIDS, Oxygen Therapies, 
etc., " Greensward Press, San Francisco, 1988, 1989, pp. 22-27.

P. Bailey et al., "Complexes and Radicals Produced During Oxonation of 
Olefins", Ozone Reactions with Organic Compounds, Advances in 
Chemistry, Series 112, pp. 1-8 (1972).


Abstract

Methods of treating systemic viral infections are disclosed comprising 
the parenteral administration of pharmacologically effective amounts 
of ozonides of terpenes in pharmaceutically acceptable carriers. 
Methods for treating viral lesions are also disclosed. In particular, 
a method for treating rfetroviral infections is disclosed. More 
particularly, a method for treating HIV infections is disclosed. In 
addition, methods for treating infections of non-retroviral viruses 
are disclosed. Further, methods for trating T-cell deficiencies are 
also disclosed. Moreover, a method of producing blood for medical 
products which is free of viral activity is disclosed.

This invention relates to methods of medical treatment. More 
particularly, it relates to the use of ozonides of terpene 
hydrocarbons in the treatment of viral infections and certain immune 
disorders.


BACKGROUND OF THE INVENTION

Methods of medical treatment employing ozonides of oil-soluble 
compounds are known in the being disclosed, for example, in U.S. Pat. 
No. 925,590 to Neel, U.S. Pat. No. 2,083,572 to McKee, and U.S. Pat. 
No. 4,451,480 to De Villez.

The prior art does not disclose the use of ozonized compounds as an 
antiviral or immunotherapeutic agent. However, particular types of 
ozonide structures have been disclosed to have certain pharmacological 
activity In U.S. Pat. No. 925,590, Neel reports the use of ozonides of 
terpenes and other ozonides for inhalation therapy, because it was 
believed to have a therapeutic effect for consumption and asthma. 
Although the Neel patent application was filed in 1902, there have 
apparently been no supporting data reported in the intervening years 
that corroborate the utility theorized by Neel.

Knox, U.S. Pat. No. 1,210,949 discloses use of ozonized castor oil as 
a laxative. Ozonation of the oil was believed to reduce its toxicity 
and create a germicidal effect.

Johnson, U.S. Pat. No. 2,356,062 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.

De Villez, U.S. Pat. Nos. 4,451,480 and 4,591,602, discloses use of 
ozonides or certain fatty acids, including olive oil, sesame oil, 
jojoba oil, castor oil and peanut oil, for external use as 
antimicrobial agents, particularly in the treatment of acne. It is 
believed that at least some of these compounds cause unacceptable skin 
irritation.

So far as can be determined, none of the medical uses of ozonides 
described in the prior art have ever been commercialized. Presumably, 
this lack of commercialization is due to unacceptable side-effects, 
toxicity, difficulties in storage, or minimal effectiveness. Many of 
these various compositions decompose on standing. Also, to the extent 
that the mechanism of action of these compositions can be attributed 
to their oxygen content, most of the ozonides known in the prior art 
have been suboptimal because these compounds typically release no more 
than about 18% of their weight as oxygen.

Methods of medical treatment employing antiviral compounds are known 
in the art. Most of the research in this area has focused on 
nucleoside analogues. Dideoxynucleosides are antiviral nucleoside 
analogues which are useful in treating retroviral infections where 
viral replication requires the transcription of viral RNA into DNA by 
viral reverse transcriptase. Other nucleoside analogues include 
deoxynucleosides and nucleoside analogues, such as acyclovir and 
gancyclovir which have only a fragment of ribose or other pentose 
connected to the base molecule. Nucleoside analogues have been shown 
to be only minimally effective in the treatment of viral infections 
that are not caused by retroviruses.

Antiviral agents other than nucleoside analogues are also known. For 
example, amantadine is an antiviral agent that prevents binding of 
certain viruses with their receptor on the cell surface. However, 
amantadine is ineffective against many known viruses.

Acquired immunodeficiency syndrome (AIDS) is a fatal condition caused 
by the human immunodeficiency virus (HIV), a retrovirus. Since AIDS 
was identified as a medical condition in 1981, over 100,000 cases have 
been reported worldwide, with over half of these cases in the United 
States. It is believed that over 2,000,000 people worldwide are 
carriers of the HIV virus, with infections continuing to spread. 
Researchers now believe that most of these carriers will one day 
develop symptoms of AIDS. No effective cure is available for AIDS, 
although dideoxynucleosides and their analogues have been shown to 
prolong life and to reduce the incidence of certain fatal infections 
associated with AIDS. Among the dideoxynucleoside analogues, AZT has 
shown the most promise as a treatment for AIDS. However, treatment of 
AIDS patients with AZT has proven to be of only poor to moderate 
effectiveness, and AZT does not cure AIDS. Moreover, in a recent human 
trial, serious toxicity was noted, evidenced by anemia (24%) and 
granulocytopenia (16%). Clearly, there is a tremendous need for a non-
toxic and effective treatment for HIV infection.

It is believed that HIV causes AIDS, in part, by infecting 
helper/inducer T4-cells and causing a T4-cell deficiency. Other 
conditions may also cause this deficiency, including immunosuppressive 
therapy for transplant patients, radio-therapy or chemotherapy in 
cancer patients, and congenital immunodeficiencies. Current immune 
boosting therapies, such as the use of interleukin-2 or .gamma.-
interferon are still in the experimental stages, and have not yet been 
proven effective. No proven effective treatments are currently in use 
for restoring a normal level of T-cells. Thus, a need exists for such 
a treatment.

Transmission of HIV through blood products has been shown to occur. 
The discovery of the HIV antibody test, and its application to blood 
products prior to release has reduced the incidence of transmission 
through blood products. However, the HIV antibody test is not 100% 
effective in detecting the presence of HIV virus particles, in part, 
because an infected individual may not produce antibodies to HIV for 
six months or longer after infection. There is, therefore, still a low 
incidence of blood products tainted with HIV being released for 
medical use. Moreover, blood products may be tainted with other 
viruses capable of being transmitted through the blood, such as 
Hepatitis B. A method of treating blood products to eliminate viral 
activity without affecting their efficacy in treatments is highly 
desireable.

Retroviruses other than HIV are known. These include the herpes family 
of viruses, HTLV I, and cytomegalovirus (CMV). Infections of these 
viruses have been notoriously difficult to treat. No vaccines are 
known for these infections. Although acyclovir has been used in the 
treatment of Herpes lesions, toxic side effects are known, and such 
treatment is not always effective. Thus, a need exists for non-toxic 
and effective treatments.

Human papilloma viruses are nonretroviral viruses responsible for 
warts of the skin or mucous membranes. Common warts are found in as 
many as 25% of some groups, and are most prevalent among children. 
Moreover, the incidence of venereal warts (condylomata acuminata and 
molluscum contageosum) has risen dramatically in the last few years, 
to the point that this condition is one of the most common sexually 
transmitted diseases in the United States. Common treatments for warts 
are often painful and invasive, and involve physical removal of the 
lesion through application of caustic agents, cryosurgery, 
electrodessication, surgical excision, or ablation with laser. 
Treatment with nucleoside analogues or interferon is also sometimes 
used. However, no treatment of proven safety and efficacy is currently 
available for warts Furthermore, at the present time, no effective 
methods of prevention are available for warts other than avoiding 
contact with infectious lesions. Therefore, a need exists for a method 
of treatment and prevention of warts.

Other nonretroviral viruses are responsible for many of the known 
infections in mammals. Vaccines are known for a minority of these 
infections. Measles, rubella, polio, rabies, certain strains of 
influenza, and mumps are examples of infections caused by viruses for 
which vaccines are known. However, the existence of a vaccine does not 
obviate the need for treatment of individuals already infected. Most 
other viruses, including Epstein Barr Virus, and most of the 
enteroviruses, reoviruses, rhabdoviruses other than rabies, 
arboviruses, and arenaviruses produce infections for which no vaccines 
are known. Currently used antiviral treatments for infections of these 
viruses include application of nucleoside analogues or amantadine, and 
various interferon treatments. Unfortunately, use of these treatments 
is of minimal or no effectiveness against infections of most of these 
viruses. The use of currently known antiviral compounds is, at best, 
moderately effective. Moreover, toxic side-effects are common. Thus a 
need exists for a wide-spectrum antiviral agent that is both non-toxic 
and effective.

Virtually all humans occasionally suffer from upper respiratory 
infections, such as colds and flu. The symptoms of these infections 
include sore throat, runny nose, itchy eyes, and earache. In addition 
to these discomforts, the infections are responsible for many days of 
absence from work and contribute to a decrease in worker efficiency. 
These infections are caused by a wide variety of viruses. Although 
vaccines are known for a minority of flu strains, no effective methods 
of prevention are known for most upper respiratory infections, and no 
truly effective methods of treatment are known for any of these 
infections. A method of treating these symptoms and underlying 
infections would be of tremendous benefit.

Moreover, there are a number of ailments that may or may not be of 
viral origin, for which no effective treatments are widely available. 
Epstein-Barr virus (EBV) is the causative agent in infectious 
mononucleosis, and has been implicated in chronic fatigue syndrome. 
Many autoimmune disorders, such as systemic lupus erythematosis and 
rheumatoid arthritis, may be associated with a virus. Whether or not 
these diseases are of viral origin, however, they are debilitating 
ailments for which an effective therapy would be of major importance.

Finally, there are a number of situations, both in research and in 
medicine, in which generation of the superoxide radical, O.sub.2.sup.-
, is advantageous. Superoxide is commonly generated through the use of 
xanthine oxidase acting on xanthine. However, these materials are 
relatively expensive and are not particularly suited for many 
utilities, including in vivo utilities. Thus, a method for generating 
superoxide that is safe and inexpensive would be advantageous.


SUMMARY OF THE INVENTION

According to the invention, there are provided novel methods of 
treatment and prevention of systemic and local viral infections. 
Methods for treating nonretroviral and retroviral infections in 
particular are also provided. More particularly, a method for treating 
retroviral HIV infections is provided. Moreover, the invention 
provides a method of treating blood products which removes any viral 
activity present in the blood. Additionally, the invention provides a 
method of treating immunosuppression characterized by T-cell 
deficiencies.

The invention, in addition, provides pharmaceutical compositions for 
use in the above novel treatments, containing ozonides of terpenes and 
a pharmaceutically acceptable carrier, and may contain other active 
ingredients. Preferably, these compositions are in dosage form 
comprising a clinically effective amount of the active compound. In 
one preferred embodiment of the invention, the pharmaceutical 
composition is comprised of an ozonized terpene in a stable injectable 
composition. In other preferred embodiments, the pharmaceutical 
compositions are in the form of nosedrops or nasal sprays, inhalants, 
throat sprays, eardrops, ophthalmic ointments or drops, vaginal or 
rectal suppositories, or ointments or creams for topical applications.
Moreover, the present invention includes the use of terpene ozonides 
and other ozonides of unsaturated hydrocarbons to treat autoimmune 
disorders, and to produce superoxide radical upon combination with an 
aqueous system.


DETAILED DESCRIPTION OF THE INVENTION

Terpene hydrocarbons are also known as isoprenoids, because they may 
generally be constructed from isoprene units. Terpene hydrocarbons are 
usually exact multiples of C.sub.5 H.sub.8. Terpenes are classified 
according to the number of isoprene units of which they are composed, 
as shown in Table 1.


 TABLE 1 

 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 may prove especially effective, when used in the method of the 
preferred embodiment, include limonene, citronella, alpha-carotene, 
beta-carotene, Vitamin A, geraniol, linalool, linalyl acetate, and 
squalene. Other compounds which are believed to make pharmacologically 
active terpene ozonides in accordance with the present invention 
include limonene, alpha-pinene, loganin, cymene, 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 ozonides in 
accordance with the present invention include labdanes, clerodanes, 
abietic acid, phyllocladene, giberellins, ophiobolin A, retigeranic 
acid, gasgardic acid, lanosterol, euphol, oleanane, ursane, lupeol, 
hydroxyhopanone, lupanes, and hopanes. Other particular terpene 
compounds which are believed to make pharmacologically active terpene 
ozonides 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 
hormone. Citral, geraniol, and nerol are particularly preferred 
terpenes. Ozonides of terpenes have three oxygen atoms replacing the 
double bonds at sites of unsaturation, creating a trioxyacyclopentane.

In the preparation of terpene ozonides, the particular desired terpene 
starting material is first obtained. A large and representative number 
of such terpenes are disclosed in the literature and/or are 
commercially available. (Many terpenes are essential oils that have 
been isolated from various parts of plants or wood by steam 
distillation or extraction.)

In the ozonide synthesis, ozone is passed through the terpene under 
conditions that provide for intimate contact between the terpene 
starting material and the ozone, such as thin film procedures, 
sparging, gas entrainment procedures, and the like. On a small scale, 
for example, the terpene is placed in a vented vessel, and ozone is 
sparged through the material until the reaction is complete. The ozone 
may advantageously be generated with any of the commercially-available 
ozone 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% O.sub.3 (ozone), with the remainder O.sub.2. This ozone mixture 
may then be sparged through the terpene at ambient temperature and 
pressure 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 
determining whether iodine gas is liberated, or by any other 
conventional technique.) Alternatively, the reaction may be followed 
by observing the weight gain of the material undergoing the reaction, 
by observing changes in physical characteristics (such as conversion 
from a liquid form to a soft paste), or by simply calculating the 
quantity of ozone needed to fully ozonate the material and stopping 
the reaction when a slight excess of ozone has passed through the 
reaction chamber. Because the reaction is exothermic, its progress may 
also be followed by monitoring the heat evolved by the reaction 
medium, and stopping the flow of ozone when the mixture ceases to 
generate heat.

When the terpene is normally a solid, such as .beta.-carotene, it may 
be solubilized in any suitable saturated nonaqueous solvent system 
prior to ozonation. With all of the terpene ozonides, it is desirable 
to exclude water, lower alcohols, nucleophilic peroxides, and proton 
donors from the reaction mixture and from the final composition, in 
order to prevent premature hydrolysis of the trioxolane ring.

Other suitable ozonation procedures may be used, such as the 
procedures disclosed in U.S. Pat. Nos. 2,083,572, 3,504,038, and 
4,451,480.

In certain preferred embodiments of the present invention, the terpene 
ozonides are formulated into pharmaceutical preparations. These 
pharmaceutical preparations include one or more terpene ozonides, and 
may further include other pharmaceutically active ingredients. In 
addition, any of the well-known pharmaceutically-acceptable carriers 
or excipients may be combined with terpene ozonides in a well-known 
manner. Suitable diluents include, for example, polyethylene glycol, 
DMSO, isopropyl myristate, and mineral oil. Conventional coloring, 
fragrance, and preserving agents may also be provided.

It is believed that the excellent weight to oxygen ratio of some of 
the terpene ozonides renders them especially effective as antiviral 
agents. Some of the terpene ozonides are capable of releasing large 
amounts of oxygen, up to 30% of the weight of the compound. This is 
because terpenes are highly unsaturated compounds. Ozonation of these 
compounds results in the addition of three oxygen atoms at each site 
of unsaturation. In addition, terpene ozonides appear to have 
significant unexpected pharmacological properties that are different 
in kind or quality from those of unrelated ozonides disclosed in the 
prior art.

The toxicity of the terpene ozonides appears to be surprisingly low in 
systemic use. Our preliminary data suggest that the LD.sub.50 for a 
representative compound, linalool ozonide, appears to be greater than 
about 5000 mg/kg in mice. Furthermore, we have discovered that the 
irritability of the terpene ozonides is surprisingly low in skin and 
eye tissues of the rabbit. It is believed that irritability of the 
compounds in humans is also surprisingly low when used in accordance 
with the methods of the preferred embodiments.

These ozonides can be used effectively in the generation of superoxide 
radical, both in and vivo. When the ozonide is combined with an 
aqueous system, gradual decomposition of the ozonide trioxolane ring 
structure occurs, with release of superoxide radical Thus, the present 
invention includes a method for generating superoxide by combining 
these ozonides with a water-containing system. For example, superoxide 
production is believed to occur when the compounds are administered to 
an organism, as well as when the compounds are mixed (with or without 
a surfactant) into a material that contains water. While the inventor 
does not wish to be limited to any particular theory of operation, it 
is believed that at least some of the beneficial and therapeutic 
properties of these ozonides are due to superoxide generation.

We have also discovered that terpene ozonides, injected in suitable 
pharmacological compositions, are effective for treatment of systemic 
viral infections. The present invention includes systemic and 
localized injection of terpene ozonides, including intravascular, 
intramuscular, subcutaneous, intraperitoneal, and other injection 
techniques. In addition, oral administration is also contemplated, 
preferably in a capsule or other nonaqueous vehicle or system.

In the method of a preferred embodiment, pharmaceutical compositions 
for systemic use such as for intravenous, intramuscular, or 
intraperitoneal injection may contain from about 0.01% to about 99% 
active ingredient, by weight. More preferred injectable compositions 
contain from about 0.05% to about 45% active ingredient, by weight. 
Moreover, pharmaceutical compositions for local application in the 
form of nosedrops or nasal sprays, inhalants, throat sprays, eardrops, 
ophthalmic ointments or drops, rectal or vaginal suppositories, or 
ointments or creams for topical applications may contain from about 
0.01% to 99.9% active ingredient, by weight. More preferred 
compositions for local application contain from about 0.05% to 50% 
active ingredient, by weight.Pharmaceutical compositions of preferred 
embodiments may contain from about 0.1% to 99.99% carrier ingredients. 
The carriers are preferably non-aqueous, because the presence of water 
rapidly leads to the degradation of the pharmacologically active 
ozonide compounds used in the preferred embodiment. The carriers 
employed in pharmaceutical compositions for systemic use are, in 
addition, preferably injectable or orally ingestible. Nonaqueous, 
injectable carriers for pharmaceutical compositions of the preferred 
embodiment for systemic application preferably include: isopropyl 
myristate, polyethylene glycol or polypropylene glycol (in liquid 
form), and DMSO, more preferably polyethylene glycol having a 
molecular weight between about 150 and 1500, most preferably about 
600. Good results have been realized, for example, by combining about 
4 parts by weight of geraniol ozonide with about 3 parts by weight 
polyethylene glycol (m.w. 600). This material is storage stable, and 
can be formulated into an injectable material by combining one part 
with three parts sterile saline immediately before use. (Although some 
superoxide production appears to begin immediately upon such 
combination, no significant degradation of the ozonide is believed to 
occur within, say, 5 minutes before the material is injected.) Another 
suitable vehicle is epal, comprising roughly equivalent parts of 
substantially anhydrous tetradecanol and dodecanol. Non-aqueous 
carriers suitable for pharmaceutical compositions for local 
application in accordance with the methods of the preferred embodiment 
include: DMSO, hydrogenated vegetable oil, mineral oil, carbomer 934, 
glycerin, propylene glycol, propyl paraben, polysorbate 60,  glyceryl 
stearate, ethanol, and modified food starch.

Therapeutic dosages of the terpene ozonides when used for systemic 
injection in accordance with the methods of the preferred embodiments 
are preferably in the range of 1 mg to 10 g active ingredient for a 70 
kg adult one time per day, more preferably in the range of 10 mg to 1 
g active ingredient for a 70 kg adult one time per day, and most 
preferably in the range of 20 mg to 500 mg active ingredient for a 70 
kg adult one or two times per day. Therapeutic dosages of the terpene 
ozonides when used for topical application in the form of creams, 
ointments, or rectal or vaginal suppositories in accordance with the 
methods of the preferred embodiments are preferably in the range of 
100 g to 10 g used one to four times per day for each cm.sup.2 of 
affected area, more preferably 1 mg to 1 g used one to four times per 
day for each cm.sup.2 of affected area, and most preferably 5 mg to 
200 g used one to four times per day for each cm.sup.2 of affected 
area. Therapeutic dosages of the terpene ozonides for use in other 
methods of local application in accordance with the preferred 
embodiments, such as nosedrops or nasal sprays, inhalants, throat 
sprays, eardrops, or ophthalmic ointments or drops are preferably in 
the range of 100 g to 1 g per application used one to four times per 
day, and more preferably 1 mg to 100 mg per application used one to 
four times per day.

Oral compositions may be given at the same dosage as the injectable 
compositions, or may be given at up to twice the injection dosage.We 
have discovered that intramuscular injection of a terpene ozonide in a 
pharmaceutically acceptable carrier, with or without contemporaneous 
oral administration, is effective in treating the symptoms of AIDS. A 
patient receiving this treatment gets fewer of the opportunistic 
infections common in AIDS patients. Such a patient also feels less 
lethargic and has a generally improved sense of physical well-being. 
This improvement in symptoms has been shown to be the result of a 
restoration of normal T4-cell levels after injection with the terpene 
ozonide. The restoration is believed to be the result of an antiviral 
effect of the terpene ozonide.

It is believed that treatment of persons infected with HIV who do not 
yet express symptoms of AIDS can be effectively treated with systemic 
injections of terpene ozonides, for example in the manner of Example 
11, in order to prevent the appearance of the symptoms of AIDS.

Furthermore, it is believed that administration of terpene ozonides in 
accordance with the present invention will be beneficial in the 
treatment of immune disorders other than AIDS. It is believed that 
systemic injection of the terpene ozonides will restore a normal level 
of T4-cells in many immunocompromised patients, including patients on 
immunosuppressive therapy, chemotherapy, or radio-therapy, and 
patients with congenital immunodeficiencies. Lupus and rheumatoid 
arthritis also respond to therapy with the terpene ozonides of the 
present invention. In a preferred embodiment, restoration of T4-cell 
levels is accomplished by systemic injection of terpene ozonides in 
the manner of Example 11.

It is also believed that treatment of blood products with a terpene 
ozonide of the present invention prior to its medical use, will 
eliminate HIV and any other viral activity present in the blood. From 
about 0.5 to about 10 mg/liter of terpene ozonide can be used in 
treating blood in, for example, a blood bank.

The terpene ozonides seem to be effective not only against HIV 
infection. They also appear to be effective in the treatment of other 
retroviral infections, such as Herpes lesions, including chicken pox, 
EBV infection, or CMV infection. Systemic injection of terpene 
ozonides is believed to be effective in treatment of these other 
retroviral infections. Additionally, topical application of the 
terpene ozonides in pharmacologically effective compositions is 
believed to be effective in the treatment of lesions of these 
retroviral infections. Moreover, it is believed that the terpene 
ozonides will be effective against many disparate viral infections, 
including viral infections of non-retroviral origin. In this regard, 
it is believed that systemic injection of a terpene ozonide in a 
pharmacologically acceptable carrier or excipient is effective in the 
treatment of systemic infections caused by non-retroviral viruses, 
including Epstein Barr Virus, most of the enteroviruses, reoviruses, 
rhabdoviruses (including rabies), arboviruses, and arenaviruses. It is 
also believed that intra-vaginal application of a terpene ozonide in a 
pharmaceutically acceptable carrier or excipient is effective against 
condylomata acuminata, molluscum contagiousum, and other viral 
infections of the vagina. Also in this regard, it is believed that 
topical application of a terpene ozonide in pharmacologically 
acceptable carrier or excipient is non-irritating and effective in the 
treatment of common warts and other viral lesions of the skin. Further 
in this regard, it is believed that application of a terpene ozonide 
in a pharmacologically acceptable carrier or excipient in the form of 
nosedrops or nasal sprays, inhalants, throat sprays, eardrops, 
ophthalmic ointments or drops, is effective in the treatment of viral 
infections of the eye, ear, nose, and throat, including upper 
respiratory infections of viral origin such as colds and flu. Finally, 
they appear to be useful in treatment of rheumatoid arthritis, which 
may be caused by a viral pathogen, as well as useful in treatment of 
other autoimmune disorders.

For example, in treating the common cold, an aerosol mist containing 2 
ml of the nasal inhalant of Example 10 may be sprayed onto each 
nostril of a patient suffering from the common cold. The process is 
repeated every four hours. Within one hour of the first treatment, the 
patient will generally report easier breathing through the nose. With 
two days of treatments, the patient can usually breathe easily through 
both nostrils and reports no sore throat.


EXAMPLE 1

Preparation of squalene ozonide
Squalene is ozonized by preparing a solution of 10 g squalene in 100 
ml hexane. Ozone gas (4% in oxygen, from a corona discharge ozone 
generator), is bubbled through this solution via a glass sparger at 
the rate of 5000 cc/min. The reaction is exothermic, and the reaction 
temperature is kept within the range of 0.degree. C. to 35.degree. C., 
preferably 20.degree. C. to 25.degree. C., and more preferably, 
22.degree. C. to 24.degree. C., using a cool water bath. The resulting 
product is the ozonide of beta carotene, and has a 98% weight gain 
over squalene.

EXAMPLE 2

Preparation of linalool ozonide
The ozonide of linalool is prepared by bubbling ozone (4% in oxygen, 
from a corona discharge ozone generator) through 100 ml neat linalool 
via a glass sparger. The reaction is exothermic, and the reaction 
temperature is kept within the range of 0.degree. C. to 35.degree. C., 
preferably 20.degree. C. to 25.degree. C., and more preferably, 
22.degree. C. to 24.degree. C., using a cool water bath. The resulting 
product is the ozonide of linalool, and has a 31% weight gain over 
linalool.

EXAMPLE 3

Preparation of Geraniol Ozonide
The ozonide of linalyl acetate was prepared by bubbling ozone (4% in 
oxygen, from a corona discharge ozone generator) through 5 ml neat 
geraniol at the rate of 5000 cc/min. The reaction mixture was cooled 
in a water bath, and after 20 minutes, the evolution of heat ceased, 
indicating completion of the ozonation process. The resulting material 
had no odor, and was soluble in polyethylene glycol (600 m.w.), 
isopropyl myristate, and mineral oil.

EXAMPLE 4

Primary skin irritation test of ozonide of linalool
Six healthy New Zealand White rabbits were tested for skin irritation. 
Approximately four hours prior to application of the ozonide 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 test site remained intact. A 1.0% solution 
of linalool ozonide in isopropyl myristate was prepared A 0.5 ml 
portion of the test solution was applied to each site by introduction 
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, 
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 Primary Irritation 
Index (PII) of 1.0. According to FHSA regulations, a material with a 
PII of less than 5.00 is generally not considered a primary irritant 
to the skin.

EXAMPLE 5

Ocular irritation test in the rabbit of the ozonide of linalool
Six healthy New Zealand White rabbits were selected for study. The 
rabbits' eyes were judged free of irritation prior to the study by 
examining with a pen light and under UV light after installation of 2% 
fluorescein stain. A 1% solution of the ozonide 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. The lids were held closed for one second. The opposite 
eye of each rabbit received 0.1 ml of the isopropyl myristate, as 
control. At 24, 48, and 72 hours post dosing, the eyes were examined 
with a pen light and re-examined with UV light following fluorescein 
staining of the cornea. Under the conditions of this test, the test 
solution was considered a non-irritant to ocular tissue of the rabbit.

EXAMPLE 6

An injectable composition for use in treatment of AIDS
250 mg/ml ozonide of geraniol from Example 3 balance oil/water 
emulsion (soybean) with 0.1% lecithin

EXAMPLE 7

A vaginal suppository for treatment of condylomata acuminata
2% w/v Ozonide of geraniol from Example 3 Balance Hydrogenated 
vegetable oil base

EXAMPLE 8

1% w/v Ozonide of linalool 60% w/v Carbomer 934  1% w/v Disodium 
edetate 10% w/v Glycerin Balance propylene
 glycol, 600 m.w.

EXAMPLE 9

A topical cream effective against chicken pox, herpes simplex and 
other viral lesions
2.5% w/v Ozonide of linalool 48% w/v Propylene glycol 30% w/v Propyl 
paraben  5% w/v Polysorbate 60 10% w/v Glyceryl monostearate Balance 
Mineral oil

EXAMPLE 10

A nasal inhalant effective against upper respiratory infections
1 mg/ml ozonide of citral balance epal 

EXAMPLE 11

Test of restoration of immune cell levels in an AIDS patient
A patient testing positive for the presence of HIV antibodies and 
diagnosed with AIDS was variously treated with the composition of 
Example 6 for a period of 99 days. On days 0 through 6, the patient 
received daily intravenous injections of 4.0 ml of the composition of 
Example 6. On days 7 to 19, the patient gas treated a.q.i.v. with the 
same composition. From days 20 through 44, the patient received no 
treatment. The patient received daily intramuscular injections from 
days 45 through 77. An immunodeficiency screening was performed on 
days 7, 20, 45, and 78. The results, expresses in cells/cmm, are shown 
in Table 2.

The results show that intra-venous injection of the composition of 
Example 6 increased the levels of all types of cells screened. These 
cell levels decreased during the period of no treatment, and remained 
relatively stable during the period of intra-muscular treatment.

It is believed that intra-venous systemic injection in the manner 
described in Example 11 is effective in the treatment of other viral 
infections as well.

EXAMPLE 12

In vitro anti-viral assay of the ozonide of linalool
A culture of SV-40 is grown in African Green Monkey (AGM) cells. The 
culture is harvested in sterile saline. The titer of SV-40 in the 
suspension is determined by Standard Plate Count Method in AGM cells. 

A working suspension of SV-40 with a titer of approximately 
1.0.times.10.sup.7 plaque forming units (PFUs)/0.1 ml is then 
prepared. Four aliquots of 1 ml each of test solution containing 2.0% 
ozonide of linalool are removed and placed in separate sterile screw-
capped tubes. Each sample is inoculated with 0.1 ml of the working 
suspension of SV-40 to yield a final concentration of approximately 
1.times.10.sup.6 PFUs/1 ml of the product. The samples are stored at 
20.degree.-25.degree. C. for a total of 28 days. Samples are selected 
at 7 day intervals to determine the number of viable PFUs present. A 
control with uninoculated solution is also stored with samples 
selected at the same intervals. At 7 days, and all subsequent sample 
selections, there are less than 10  PFUs present. No PFUs are present 
in any control sample.

EXAMPLE 13

Preparation of blood products free of viral activity
Blood obtained from a donor is mixed with 0.5 g of ozonide of geraniol 
from Example 3 per unit (500 ml) of blood. The blood is then processed 
in the normal manner. The resulting blood products are free of 
detectable HIV or other viral activity using standard viral assays.

EXAMPLE 14

Test for efficacy of treatment of chicken pox
A small dose (approximately 25 l) of the composition of Example 9 is 
topically applied to each lesion on the left side of a child suffering 
from chicken pox. Lesions on the right side are treated with the 
composition lacking in active ingredient. Within 24 hours, the lesions 
on the child's left side are significantly reduced with little or no 
self-induced trauma from scratching. The lesions on the child's right 
side are unchanged in size, and show the effects of trauma from 
scratching.

In a manner similar to that employed in Example 15, other viral 
lesions, such as common warts and herpes lesions may be treated by 
topical application of a terpene ozonide in a pharmaceutically 
acceptable carrier or excipient.

EXAMPLE 15

Test for efficacy of treatment of condylomata acuminata
A 5 ml suppository with the composition of Example 4 is administered 
intra-vaginally to one group of patients suffering from condylomata 
acuminata. A second group of such patients receive a suppository 
without the active ingredient of Example 4. A third group receives 
cryogenic treatment of the affected area, a commonly used treatment 
for condylomata acuminata. The average size of the lesions in each 
group is approximately 2 cm.sup.2. Within seven days, the patients of 
the first group have reduced reddening of the vagina and within 15 
days, colposcopy does not reveal papilloma viruses. In the second 
group of patients, the lesions are unchanged after 15 days. Patients 
in the third group have no condylomata lesions immediately after 
treatment, however, these patients continue to complain of pain and 
bleeding for up to 30 days after the procedure is performed.

EXAMPLE 16

Treatment of Rheumatoid Arthritis
It has been theorized that rheumatoid arthritis is caused by a viral 
agent. The antiviral ozonides of the present invention are believed to 
be efficacious in treatment of this disease. Thus, a 20% oral 
preparation comprising capsules containing citral ozonide in medium 
chain triglyceride (MCT) is prepared and is taken twice daily by a 
patient suffering from rheumatoid arthritis. Each dose delivers 400 mg 
active ingredient to the 60 kg patient. After 1 week, the ANA of the 
patient has dropped from approximately 2500 to 100, indicating 
remission of the disease. Similar treatment is effective against 
psoriasis.


Claims

I claim:

1. A method for treating viral infections of HIV in a mammal, 
   comprising the parenteral application of a pharmacologically 
   antiviral effective amount of an ozonide of a terpene in a 
   pharmaceutically acceptable carrier or excipient.
2. The method of claim 1, wherein said terpene is selected from the 
   group consisting of: limonene, citronella, alpha-carotene, beta-
   carotene, vitamin A, linalool, linalyl acetate, squalene, geraniol, 
   alpha-pinene, loganin, cymene, farnesanes, eudesmanes, acoranes, 
   cedranes, chamigranes, caryophyllanes, illudanes, humulenes, 
   himachalenes, longifolanes, perhydroazulenes, quaianes, 
   quaianolides, germacranes, labdane, clerodanes, abietic acid, 
   phyllocladene, giberellins, ophiobolin A, retigeranic acid, 
   gasgardic acid, lanosterol, euphol, oleanane, ursane, lupeol, 
   hydroxyhopanone, lupanes, hopanes, B-selinene, zingibene, camphene, 
   sabinene, ocimene, myrcene, nerol, citral A, citral B, farnesol, 
   bisabolene, phytol and cecropia juvenile hormone.
3. The method of claim 1, wherein said terpene is a hemi-terpene,
   mono-terpene, sesqui-terpene, di-terpene, ses-terpene, tri-terpene, 
   or tetra-terpene.
4. The method of claim 1, wherein said viral infection is systemic, 
   said parenteral application is by systemic injection, and said 
   composition is for systemic injection, and comprises an injectable 
   non-aqueous carrier.
5. The method of claim 1, wherein said viral infection produces local 
   sites of infection.
6. The method of claim 5, wherein said viral infection is an upper
   respiratory infection.
7. The method of claim 5, wherein said viral infection is an infection 
   of the eye, ear, nose, or throat.
8. A method of treating mammalian blood to be used for medical 
   applications in the treatment of HIV comprising the addition of an 
   azonide of a hemi-terpene, mono-terpene, sesqui-terpene, di-
   terpene, ses-terpene, tri-terpene, or tetra-terpene to said blood 
   causing said blood to be free of viral activity.