KR20070023721A - Use of targeted oxidative therapeutic formulation in bone regeneration - Google Patents

Abstract

The present invention relates to pharmaceutical formulations and their use. Such pharmaceutical formulations may include reaction products or peroxide species resulting from the oxidation of alkenes such as geraniol with an oxygen-containing oxidizing agent such as ozone; Permeable solvents such as dimethylsulfoxide ("DMSO"); Dyes comprising chelated metals such as hematoporphyrin; And aromatic redox compounds, such as benzoquinone. The pharmaceutical formulation is used to effectively stimulate bone regeneration and increase osteoblast activity in the patient.

Bone regeneration, osteoblasts, oxidative therapeutic formulations, oxygen-containing oxidants

Description

Use of targeted oxidative therapeutic formulation in bone regeneration

This application claims priority to US Provisional Patent Application No. 60 / 569,554, filed on May 10, 2004, entitled "Use of Targeted Oxidative Therapeutic Formulations in Bone Regeneration," and is herein incorporated by reference. The entirety of which is incorporated by reference.

The present invention relates to compositions containing peroxide species or oxidation products, methods for their preparation and their use. More specifically, the present invention relates to a peroxide species or reaction product produced by oxidizing an olefinic compound in liquid form or solution with an oxygen-containing oxidant; Permeable solvents; Dyes containing chelated metals; And it relates to a pharmaceutical composition or formulation comprising an aromatic redox compound. The present invention also relates to the manufacture of pharmaceutical formulations and their use in bone regeneration.

Musculoskeletal and limb trauma are a difficult economic burden in both developing and developing countries. As life expectancy increases and the number of older populations increases, the incidence of musculoskeletal diseases is increasing worldwide. In the United States alone, estimates of the total cost associated with musculoskeletal pathology amount to $ 250 billion annually (World Health Organization, 2000). As the number of people over age 65 increases, the total annual medical costs for these injuries will continue to increase. Direct burdens include expenses and fees for hospital and nursing home care, physicians and other professional services, rehabilitation, community-based services, use of medical equipment, prescription drugs, local rehabilitation, home renovation, and insurance management. Direct costs are not responsible for the long-term consequences of these injuries, such as disability, reduced productivity, lost wages, and reduced quality of life.

In addition, current techniques and methods for promoting bone repair and implant fixation suffer from significant drawbacks. Traditionally applied bone cements and sealers can adversely affect the repair of osteoblasts. Studies have shown that root canal sutures interfere with the healing process around the root roots by inhibiting osteoblast proliferation (Granchi, et al., 1995). In addition, despite its excellent biocompatibility, hydroxyapatite appears to delay bone formation due to its physical presence (Beck-Coon, et al., 1991). In addition, macrophages and osteoblasts react adversely to metals such as aluminum, lead and cadmium.

Chronic dental infections and root canal procedures may also be risk factors in the development of distal pathological conditions, such as atherosclerosis. Despite antibiotics, root canal therapy and surgical discharge, complete eradication of anaerobic infectious diseases of the periodontal bone is difficult. Closure of the infected "dead space" by excretion, macrophage initiation and osteoblast repair of osteoblasts all need to occur to prevent the lesion from distal infection.

The function of macrophages and osteoblasts depends on precisely acting intracellular relationships between mitochondria, microfibers and peroxidation chemistry. Mitochondria are also important players in the calcium dynamics of cells and regulate the supply of release-capable secretory granules (Chakraborti, et al., 1999). Evidence is presented that osteoblasts retain calcium phosphate in the form of granules in their mitochondria (Plachot, et al., 1986). In addition, induction of osteoblast function in bone repair appears to require adequate mitochondrial outer membrane function. Intracellularly regulated peroxidation is a known inducer of osteoblast turnover and calcium secretion in bone repair.

Ozone is a triatomic gas molecule and is the allotrope of oxygen. Ozone can be obtained through pure oxygen as an electrical discharge or strong ultraviolet light. The popular misconception that ozone is a serious pollutant, the "free radical" theory, and the market for antioxidant supplies has led to widespread prejudice of medical theories against ozone's therapeutic uses. However, ozone therapy is a wrong name. Ozone is an extremely reactive, unstable gas with a mechanism of action that is directly related to by-products produced through selective interaction with organic compounds present in plasma and in cell membranes. The selective reaction of unsaturated olefins with ozone occurs at carbon-carbon double bonds, producing ozonide. Ozone is toxic in itself, and its reaction product, ozonide, is unstable and, by itself, does not help in treatment.

Hydrogen peroxide (H ₂ 0 ₂ ), discovered in 1818, is present in trace amounts in nature. Hydrogen peroxide is unstable and volatilizes and decomposes (or foams) when in direct contact with organic membranes and particulate matter. Light, stirring, heating and iron all accelerate the rate of hydrogen peroxide decomposition in solution. Hydrogen peroxide kills microorganisms with low levels of peroxide-destroying enzymes such as catalase by in vitro direct contact. However, when hydrogen peroxide is injected into the blood of rabbits infected with peroxide-sensitive E. coli , there is no bactericidal effect. In addition, increasing the ex vivo peroxide concentration in rabbit or human blood containing this coli showed no evidence of direct bactericidal activity. The lack of high concentrations of hydrogen peroxide is directly related to the presence of peroxide-destroying enzyme catalase in the host animal blood. To have an effect, high concentrations of hydrogen peroxide must be contacted with bacteria for a significant period of time. In general, large amounts of hydrogen peroxide-depleting enzymes, such as catalase, present in the blood prevent peroxides from being present in the blood for more than a few seconds. Thus, hydrogen peroxide introduced into the bloodstream by injection or infusion does not act directly as an extracellular fungicide in blood or extracellular fluid.

However, hydrogen peroxide participates in the sterilization process of activated macrophages. Activated macrophages are attracted to the site of infection, attached to the infected organism, and consumed. The death of the organism is caused by hydrogen peroxide in the macrophages. Hydrogen peroxide oxidizes the cell's chlorides with chlorine dioxide free radicals, which destabilize the microbial membrane and, if persistent, induce apoptosis or cell suicide. An important therapeutic criterion for intracellular peroxidation is the selective delivery, uptake and activation of peroxide transport molecules only to diseased macrophages, which are believed not to enhance catalase and glutathione reductase activity. Infused hydrogen peroxide is a generalized poison, but targeted intracellular peroxidation is an optional therapeutic tool.

Macrophages play an important role in immunity, bone calcification, vision, nerve isolation (follicle formation), detoxification, pumping intensity and removal of toxins from the body, depending on their presence. The energy requirements of macrophages are met by intracellular structures called mitochondria. Mitochondria are often structurally involved in microfiber inner cell construction. The folded inner layer of mitochondria produces high energy molecules ATP and the outer layer contains electron recycling molecules that produce cytochromes and peroxides. The outer layers of mitochondria are susceptible to toxic blockage or damage by endotoxins, mycotoxins, viruses encoded toxins, drugs, heavy metals and pesticides. When the peroxidation of mitochondria is blocked, the filamentary structure of the cells tends to crosslink and cause inaccurate signals, failure, inadequate replication or early cell death.

US Patent No. 4,451,480 to De Villez teaches compositions for treating acne and methods thereof. This method involves the treatment of the site of infection with ozonation materials typically induced by ozonation of various fixed oils and unsaturated esters, alcohols, ethers and fatty acids.

US Pat. No. 4,591,602 to De Villez presents Jojoba's ozonide used to control microbial infections.

US Pat. No. 4,983,637 to Herman describes a method for parenteral treatment of local and systemic viral infections by administering an ozonide of terpene in a pharmaceutically acceptable carrier.

US Pat. No. 5,086,076 to Herman discloses an antiviral composition containing an ozonide of a carrier and a terpene. The composition is suitable for systemic administration or topical application.

US Pat. No. 5,126,376 (Herman) describes a method for topically treating viral infections in a mammal using an ozonide of terpene in a carrier.

US Pat. No. 5,190,977 (Herman) teaches antiviral compositions containing the ozonide of terpenes and non-aqueous carriers suitable for systemic injection.

US Pat. No. 5,190,979 to Herman describes a method for parenteral treatment of a medical condition in a mammal using an ozonide of terpene in a carrier.

US Pat. No. 5,260,342 (Herman) teaches a method for parenteral treatment of a viral infection in a mammal using an ozonide of terpene in a carrier.

US Pat. No. 5,270,344 to Herman discloses a method of treating systemic diseases in a mammal by applying a diperoxide derivative of trioxolane or an unsaturated hydrocarbon to the intestine of a mammal, wherein the derivative is dissolved in a nonpolar solvent. Prepared by ozone decomposition of unsaturated hydrocarbons.

US Pat. No. 5,364,879 to Herman describes a composition for treating a medical condition in a mammal, wherein the composition comprises a trioxolane derivative of a diperoxide or non-terpene unsaturated hydrocarbon, the derivative being in a carrier Unsaturated hydrocarbons are prepared by ozone decomposition at 35 ° C or below.

Although the use of terpene ozonide for different medical indications has been reported, terpene ozonide exhibits several deficiencies. For example, ozonide of monoterpenes, such as myrcene and limonene, are flammable in the laboratory. As a result, this is very dangerous to manufacture or store.

Thus, there is a need for a safe and effective pharmaceutical formulation or composition that utilizes a reaction product produced by oxidizing an alkene compound. There is also a need for methods to stimulate mitochondrial defense against free radical formation and to effectively treat individuals with cancer such as lymphoma.

summary

The present invention provides peroxide species or reaction products resulting from the oxidation of unsaturated organic compounds in liquid form or solution with oxygen-containing oxidants; Permeable solvents; Chelated dyes; And an aromatic redox compound. In one embodiment of the pharmaceutical formulation, the essential ingredients include peroxidation products, stabilizing solvents, metalloporphyrins and quinones produced by ozone decomposition of unsaturated alcohols. The invention also relates to the use of pharmaceutical formulations for treating cancer.

Peroxide species or reaction products are preferably formed through the reaction of alkenes and ozone. The reaction between alkenes and ozone is generally driven by a Criegee mechanism. According to this mechanism, as can be seen in Scheme 1 below, in the initial stage of the reaction, ozone is reacted with alkenes to a 1,3-bipolar addition ring reaction to give the first ozonide (1,2,3-trioxalane) To obtain. The primary ozonide is unstable and undergoes 1,3-cycloreversion to carbonyl compounds and carbonyl oxides. In the absence of other reagents or nucleophilic solvents, these novel 1,3-dipoles are introduced in a second 1,3-bipolar cyclocycle to yield "standard" ozonide, 1,2,4-trioxalane. Create

In the side reactions, as can be seen in Scheme 2 below, carbonyl oxide can be introduced into the dimerization to produce the peroxide dimer, 1,2,4,5-tetraoxane.

Carbonyl oxide is a strong electrophilic species and in the presence of nucleophilic species (eg alcohol or water), nucleophilic addition reactions readily produce 1-alkoxyhydroperoxide, as shown in Scheme 3. Under certain conditions, 1-alkoxyhydroperoxide may be further reacted to produce carboxylic acid derivatives.

Again, without being bound by theory, it is expected that in the present invention three major forms of peroxidation products, i.e., standard ozonide, carbonyl tetraoxane dimer and 1-alkoxyhydroperoxide, exist during ozonation of alcohol-containing alkenes. It is reasonable. In the presence of water, some of these peroxide products may also allow the presence of organic peracids in the crude product mixture.

The present invention also relates to the use of a permeable solvent such as dimethylsulfoxide ("DMSO") to "stabilize" the initial product of ozone decomposition. Similarly, without being bound by theory, stabilization is considered to be the most likely simple solvation phenomenon. However, DMSO is originally known as a nucleophile. It may also participate as a nucleophilic partner (eg, as a dimethylsulfonium salt) in stabilizing reactive species. Stabilized peroxide molecules and permeable solvents of the pharmaceutical formulations are prepared from components that are generally considered to be safe (“GRAS”: Generally Regarded As Safe).

Another component of the pharmaceutical formulation is a chelated dye such as porphyrin. The tendency of metalloporphyrins to sensitize oxygen under photochemical excitation is documented in detail, as is the tendency for ferroporphyrin and copper porphyrin to bind oxygen-containing systems.

Further components of the pharmaceutical formulation are aromatic redox compounds, such as quinones.

Without being bound by theory, it is claimed that preferred pharmaceutical formulations are combinations of biochemical agents that induce regenerative autocatalytic oxidation in infected macrophages or dysplastic macrophages. Pharmaceutical formulations stimulate targeted apoptosis (apoptosis) through uninterrupted peroxidation. Thus, pharmaceutical formulations exert a therapeutic effect on a number of apparently different mitochondrial-based macrophage disease. In particular, it has been found that pharmaceutical formulations promote bone re-growth and healing of periapical tooth abscesses, periodontal lesions, and complex fractures. The pharmaceutical formulation is effective for stimulating osteoblast regeneration activity. These results suggest its effect on bone regeneration.

The present invention provides peroxide species or reaction products resulting from the oxidation of unsaturated organic compounds in liquid form or solution with oxygen-containing oxidants; Permeable solvents; Chelating dyes; And an aromatic redox compound. The pharmaceutical formulations can be used to stimulate bone regeneration and osteoblast activity in patients with bone injury such as abscesses, lesions, fibromas and fractures. In one embodiment of the present invention, the essential components of the pharmaceutical formulation include peroxidation products, stabilizing solvents, metalloporphyrins and quinones which are produced by ozonating unsaturated alcohols.

The unsaturated organic compound of the pharmaceutical formulation, which may be an unsaturated olefinic hydrocarbon, may be a hydroxyl group-free alkene or a hydroxyl-containing alkene. Preferably, the alkenes have less than about 35 carbon atoms. The hydroxyl group-free alkene group can be an open-chain unsaturated hydrocarbon, monocyclic unsaturated hydrocarbon or bicyclic unsaturated hydrocarbon. The hydroxyl-containing alkenes may be open-chain unsaturated alcohols, monocyclic unsaturated alcohols or bicyclic unsaturated alcohols. Alkenes may also be contained in fixed oils, esters, fatty acids or ethers.

The unsaturated olefinic hydrocarbons that can be used may be unsubstituted, substituted, cyclic or complexed alkenes, hydrazines, isoprenoids, steroids, quinolines, carotenoids, tocopherols, prenylated proteins or unsaturated fats. Preferred unsaturated hydrocarbons of the present invention are alkenes and isoprenoids.

Isoprenoids are found primarily in plants as components of essential oils. Although many isoprenoids are hydrocarbons, oxygen-containing isoprenoids are also present, for example, as alcohols, aldehydes and ketones. In general terms, isoprene itself is known to be the end product of isoprenoid biosynthesis and is not an intermediate, although the components of isoprenoid hydrocarbons are to be understood as hydrocarbon isoprene, CH ₂ = C (CH ₃ ) -CH = CH ₂ . Can be. Isoprenoid hydrocarbons are classified by the number of isoprene (C ₅ H ₈ ) units containing them. Thus, there are two monoterpenes, three sesquiterpenes, four diterpenes, five seterterpenes, six triterpenes, and eight isoprene units for tetraterpenes, respectively. Tetraterpenes are more commonly known as carotenoids.

Limonene and pinene are examples of monoterpenes. Farnesol and nerolidol are examples of sesquiterpene alcohols. Vitamin A ₁ and phytol are examples of diterpene alcohols and squalene is an example of triterpenes. Provitamin A ₁ , known as carotene, is an example of tetraterpene. Geraniol, monoterpene alcohols are liquid in oxygen bound and standard conditions and are safe for living cells.

Preferred unsaturated hydrocarbons for pharmaceutical formulations include alkene isoprenoids, for example myricene, citylene, citral, pinene or limonene. Preferred unsaturated hydrocarbons also include linear isoprenoid alcohols having 2 to 4 repeating isoprene groups which are straight chains, for example terpineol, citronellol, nerol, phytol, menthol, geraniol, geranyl Raniol, linalool or farnesol.

Unsaturated organic compounds can be straight, branched, cyclic, helical or complexed with other molecules in their configuration. Unsaturated organic compounds may exist in their natural gaseous liquid or solid state prior to binding to the oxidant.

Open-chain unsaturated hydrocarbons include C _n H _2n , 1 double bond, n = 2-20; C _n H _{2n -2} , two double bonds, n = 4-20; C _n H _{2n -4} , three double bonds, n = 6-20; C _n H _{2n- 6} , four double bonds, n = 8-20; C ₂₅ H ₄₀ , seterterpene hydrocarbons; Or C ₃₀ H ₄₈ , triterpene hydrocarbons.

Monocyclic unsaturated hydrocarbons include C _n H _{2n -2} , 1 double bond and 1 ring, n = 3-20; C _n H _{2n -4} , two double bonds and one ring, n = 5-20; C _n H _{2n- 6} , three double bonds and one ring, n = 7-20; C ₂₅ H ₄₀ , seterterpene hydrocarbons; Or C ₃₀ H ₄₈ , triterpene hydrocarbons.

Bicyclic unsaturated hydrocarbons include C _n H _{2n -4} , one double bond and two rings, n = 4-20; C _n H _{2n- 6} , two double bonds and two rings, n = 6-20; C ₂₅ H ₄₀ , seterterpene hydrocarbons; Or C ₃₀ H ₄₈ , triterpene hydrocarbons.

Open-chain unsaturated alcohols include C _n H _2n O _m , 1 double bond, n = 3-20, m = 1-4; C _n H _{2n 2} O _m , two double bonds, n = 5-20, m = 1-4; C _n H _{2n -4} O _m , 3 double bonds, n = 7-20, m = 1-4; C _n H _{2n- 6} O _m , four double bonds, n = 9-20, m = 1-4; C ₂₅ H ₄₀ O _m , m = 1-4, sesterterpene alcohol; Or C ₃₀ H ₄₈ O _m , m = 1-4, triterpene alcohol.

Monocyclic unsaturated alcohols include C _n H _{2n 2} O _m , 1 double bond and 1 ring, n = 3-20, m = l-4; C _n H _{2n -4} O _m , two double bonds and one ring, n = 5-20, m = 1-4; C _n H _{2n- 6} O _m , 3 double bonds + 1 ring, n = 7-20, m = 1-4; C ₂₅ H ₄₀ O _m , m = 1-4, sesterterpene alcohol; Or C ₃₀ H ₄₈ O _m , m = 1-4, triterpene alcohol.

Bicyclic unsaturated alcohols include C _n H _{2n -4} O _m , one double bond and two rings, n = 5-20, m = 1-4; C _n H _{2n- 6} O _m , two double bonds and two rings, n = 7-20, m = 1-4; C ₂₆ H ₄₀ O _m , m = 1-4, sesterterpene alcohol; Or C ₃₀ H ₄₈ O _m , m = 1-4, triterpene alcohol.

Based on the total weight of the pharmaceutical formulation, the alkenes may vary from about 0.001 to about 30%, preferably from about 0.1 to about 5.0%, more preferably from about 0.5 to about 3.0%.

Oxygen-containing oxidizing agents in pharmaceutical formulations that oxidize unsaturated hydrocarbons are singlet oxygen, triplet oxygen, superoxide anions, ozone, periodate, hydroxyl radicals, hydrogen peroxide, alkyl peroxides, Carbamyl peroxide, benzoyl peroxide or oxygen bound to a transition element, for example molybdenum (eg M ₀ O ₅ ).

A preferred method of incorporating "active oxygen" in intact isoprenoid alcohols, such as geraniol, is to ozonate in the dark in the absence of water or polar solvents at temperatures between 0 and 20 ° C. Geraniol “ozonide” is then dissolved in the dark in 100% DMSO and stabilized to prevent premature degradation of the product. Without being bound by theory, it is believed that the catalytic decomposition of tetraoxane peroxide dimer byproducts of geraniol ozonation, but not ozonide, takes place intracellularly in the presence of superoxide anions. Final reactive therapeutic agents released are hydrogen peroxide and acetic acid.

Pharmaceutical formulations also utilize permeable solvents. Permeable solvents that stabilize oxygen-bonded unsaturated hydrocarbons can be emollients, liquids, liposomes, micelle membranes or vapors. Permeable solvents that can be used include aqueous solutions, fats, sterols, lecithin, phosphides, ethanol, propylene glycol, methylsulfonylmethane, polyvinylpyrrolidone, pH-buffered saline and dimethylsulfoxide ("DMSO"). . Preferred permeable solvents include DMSO, polyvinylpyrrolidone and pH-buffered saline. The most preferred permeable solvent is DMSO.

Based on the total weight of the pharmaceutical formulation, the permeable solvent may vary from about 50 to about 99%, preferably from about 90 to about 98%, more preferably from about 95 to about 98%.

"Stable" peroxide molecules and their permeable solvents have been prepared from components currently used in the manufacturing regulated by the Food and Drug Administration ("FDA"). These ingredients are subject to Drug Master Files, Drug Monographs, found in USP / NF, or generally recognized as safe ("GRAS").

Another component of the pharmaceutical formulation is a chelated dye. The dyes preferably comprise chelated divalent or trivalent metals such as iron, copper, manganese, tin, magnesium or strontium. Preferred chelating metal is iron. The tendency of chelated dyes, such as metalloporphyrins, to sensitize oxygen under photochemical excitation is documented in detail, as is the tendency for ferroporphyrin and copper porphyrin to bind to oxygen-containing systems. Dyes that can be used include natural or synthetic dyes. Examples of these dyes include porphyrin, rose bengal, chlorophyllin, hemin, porphine, corinine, texaprine, methylene blue, hematoxylin, eosin, erythrosin, flavinoids, lactoflavin, anthracene dyes, hypericin, Methylcolanthrene, neutral red, phthalocyanine, fluorescein, eumelanin and feomelanin. Preferred dyes may be any natural or synthetic porphyrin, hematoporphyrin, chlorophyllin, rose bengal, each homolog thereof or mixtures thereof. Most preferred dyes are a mixture of hematoporphyrin and rose bengal, or a mixture of hematoporphyrin and chlorophyllin. The dye may be reactive to photons, lasers, ionizing radiation, negatives, electric heartbeats, electroporation, magnetic pulses or continuous flow excitation.

Based on the total weight of the pharmaceutical formulation or composition, the dye may vary from about 0.1 to about 30%, preferably from about 0.5 to about 5%, more preferably from about 0.8 to about 1.5%.

Further components of the pharmaceutical formulation are aromatic redox compounds, such as quinones. The aromatic redox compound may be all substituted or unsubstituted benzoquinone, naphthoquinone or antroquinone. Preferred aromatic redox compounds include benzoquinone, methyl-benzoquinone, naphthoquinone and methyl-naphthoquinone. Most preferred aromatic redox compounds are methyl-naphthoquinones.

Based on the total weight of the pharmaceutical formulation, the aromatic redox compound may vary from about 0.01 to about 20.0%, preferably from about 0.1 to about 10%, more preferably from about 0.1 to about 0.5%.

The pharmaceutical formulation may also be activated by an energy source or electron donor. Useful electron donors include NADH, NADPH, current, ascorbate or ascorbic acid and germanium sesquioxide. Preferred electron donors include ascorbate and germanium sesquioxide. Most preferred electron donor is ascorbic acid in any salt form.

Based on the total weight of the pharmaceutical formulation, the electron donor can vary from about 0.01 to about 20%, preferably from about 1 to about 10%, more preferably from about 1 to about 5%.

In order to obtain a biological effect in vivo, the pharmaceutical formulation is preferably injected with the superoxide-producing chelated dye and aromatic quinone, as an ozonation-produced peroxide product of unsaturated hydrocarbons, rather than ozonide. Unsaturated hydrocarbon products or peroxide dimer molecules can be stabilized in non-aqueous stabilizing solvents and can penetrate the lipid membrane.

Researchers of vigorously activated dye treatments have long known that superoxide producing dyes and aromatic redox compounds are preferentially absorbed into infected dysplastic cells of catalase deficiency. Without wishing to be bound by theory, catalase-induced disruption of peroxides can be inhibited either naturally in the target cell or by pharmaceutical formulation. The peroxide dimer must also be activated by a superoxide producing dye which initiates electron donation to the dimer and causes the release of hydrogen peroxide and acetic acid in the cell. Electronic activation of the dye does not always require light, but rather can occur through small electrical pulses provided by, for example, heart rate. Accordingly, peroxidation reactions in infected macrophages tend to disrupt the prenylated protein binding of microtubules, destroy infectious toxins, or induce apoptosis of macrophage host cells in cells.

Pharmaceutical formulations are combinations of stable ingredients. These components can preferably be stored in separate containers as anhydrous solid components and liquid components, which are then mixed in place for use. Preferably, the anhydrous solid component comprises chelated dyes and aromatic redox compounds. Preferably, the liquid component comprises a peroxide species or reaction product, resulting from the oxidation of unsaturated hydrocarbons with an oxygen-containing activator, with a permeable solvent. Preferably it is administered intravenously. Preferably, the reconstituted product may be administered intravenously as a concentrate diluted in saline. Endodontic, direct intraosseous, intraarterial, topical, intraperitoneal and intradural delivery is also possible via the route of administration. Intramuscular injections are undesirable because they tend to cause local irritation.

In vivo administration of the pharmaceutical formulations is effective in treating viruses and symptoms of viruses in infected patients. In particular, the pharmaceutical formulation stimulates promoted healing and bone re-growth in tooth abscesses, bone lesions and fractures. The pharmaceutical formulations increase osteoblast activity and are effective in bone regeneration.

Example  1: Ozone Decomposition of Unsaturated Hydrocarbons

Ozonation of alkenes can be carried out in or without solvent. In each case, cooling of the reaction mixture is important to avoid explosive decomposition of the peroxide product of the reaction.

The following general method is common in ozone decomposition of liquid alkenes.

The 1 L flask equipped with a magnetic stirrer was filled with alkene (2 mol) and the apparatus was weighed. Surround the flask with a cold bath (ice water or ice-salt). Once the contents cooled to below 5 ° C., stirring was started and a stream of ozone in anhydrous oxygen (typically 3% ozone) was passed through the mixture. It is advantageous to disperse the ozonated oxygen through the glass frit, but this is not necessary in the stirred solution. Periodically, the gas stream was stopped and the reaction flask was weighed or the reaction mixture sampled. Then the gas stream was restarted.

If the mass of the reaction flask shows a sufficient weight increase, or if the proton magnetic resonance ("H ¹ NMR") spectrum of the reaction mixture shows the desired decrease in the intensity of the olefinic proton resonance (usually about 50%), stop the gas flow. Let's do it.

Ozone decomposition can be carried out as described above by replacing a solution of alkenes in a solvent that is non-reactive with ozone, such as saturated or chlorinated hydrocarbons. Ozonation can also be carried out using alkenol instead of alkenes, as described above, without affecting the reaction in a substantial manner in the presence or absence of a solvent.

The reaction mixture is then poured slowly into the cooled permeable solvent.

Example  2: Preparation of Pharmaceutical Formulations

Preferred pharmaceutical formulations of the invention were prepared as follows:

(1) 120 mg / L of an ozone / pure oxygen gas mixture is sparged with 1 liter gas per hour through alkadiene alcohol, 3,7-dimethyl-2,6-octadiene-1-ol (geraniol);

(2) maintaining the reaction at about 5 ° C .;

(3) removing a small aliquot of the reaction product per hour and determining the formation of peroxide species or reaction product by H ¹ NMR;

(4) stopping the reaction when at least about 50% of the available unsaturated bonds have reacted;

(5) the product mixture was diluted with dimethylsulfoxide (1:10) to give a solution or dispersion;

(6) Prior to use in the target biological system, when the target biological system is delivered by intravenous saline infusion, a mixture of hematoporphyrin, rose bengal, and methyl-naphthoquinone anhydrous powder, It was added to the solution or dispersion in an amount sufficient to produce 20 μmol. Ascorbate may optionally be added to the formulation prior to use.

Example  3: Examples of Pharmaceutical Formulations

Two preferred formulations are as follows:

A.

weight% ingredient 0.54 * Tetraoxane Dimer of Acetal Peroxide from Ozonation of Geraniol 98.00 DMSO 0.83 Hematoporphyrin 0.24 Methylnaphthoquinone 0.39 Rose Bengal

* Measured by mass spectrometer.

B.

weight% ingredient 0.54 * Tetraoxane Dimer of Acetal Peroxide from Ozonation of Geraniol 98.00 DMSO 0.83 Hematoporphyrin 0.24 Methylnaphthoquinone 0.39 Chlorophyllin Sodium-Copper Salts

* Measured by mass spectrometer.

Example  4: Inside ( endodontic Treatment of bone abscess

Fifteen patients with periapical bone abscess participated in this study to determine the effect of pharmaceutical formulation on bone repair and healing after endodontic treatment. Twelve patients were treated with augmented intradermal necrosis as an example of a pharmaceutical formulation. Three were used as normal controls without treatment. Infected teeth of treated patients were opened and necrotic tissue removed. An example of a pharmaceutical formulation having Formulation A from Example 3 above was injected into the canal and periapical bone with 0.2 ml Ca (OH) _{2 at} a concentration of 1 cc pharmaceutical formulation to 10 cc saline. Radiographic recordings of the periapical abscess were performed 6-8 weeks after intradental treatment. Controls were treated with non-augmented intradental necrosis. After removal of necrotic tissue, closure, gutta percha tube-filling, and intervening prosthetic ("IRM") sealing were performed in both treated and control patients.

Treated Subject

The 79 year old male had a history of infected fistula penetrating the lateral gingival area of the tooth (position 2) for 1 month. Early radiographs showed extensive abscesses in the periapical bones on the teeth. The subject underwent dental surgery and released a purulent exudate from the root canal and fistula. Teeth and bones were initially treated with enhanced pulp necrosis. Secondary necrosis removal and crown filling with beat Perca were performed after 3 weeks. After 14 days, the fistula was resolved and subsequent radiographs showed rapid dissipation of the periapical abscess with rapid bone regrowth.

A 50-year-old male had a history of cold-sensitive susceptibility to the teeth (position 19) for 1 month. The teeth had already been treated intradermally 10 years ago with partial filling of the root canal. Early radiographs showed some degree of periapical radiographs around the anterior muscles. The patient was re-operated with enhanced endodontic surgery as described above. All symptoms resolved within 48 hours. The endodontic procedure was completed after 3 weeks with beat Perca filling. Subsequent radiographs showed that near periapical radiopacity nearly resolved with increased bone density.

The 43-year-old woman had a history of facial swelling and pain according to percussion susceptibility in the teeth (position 6) for 2 months. Early radiographs showed the periapical radiographs around the root of the tooth. The patient underwent enhanced endodontic surgery as described above. Facial swelling resolved within two weeks. The endodontic procedure was completed after 3 weeks using a beater Perca filling. Subsequent radiographs showed that near periapical radiopacity nearly resolved with increased bone density.

A 50 year old woman had facial swelling pain on her teeth (position 19). The teeth received intradermal treatment three months ago but were not successful. The patient also received radioactive iodine treatment for thyroid cancer six months ago. Early radiographs showed previous incomplete filling and extensive periapical radiographs near the dorsal roots. The patient underwent enhanced endodontic surgery as described above. Bacterial culture of purulent by-products showed Neisseria and Alpha Streptococcus . After treatment, facial swelling resolved within 4 days. The endodontic procedure was completed 6 weeks later with a beaten Perica filling. Subsequent radiographs showed that the increase in bone density and near periapical radiographs were almost resolved.

A 59-year-old woman had a history of fistula over the palatal proximal region of the teeth (position 2). The patient had periodontal surgery at that site four months ago. Cultivation of by-products removed from the fistula is Beta Streptococcus , Enterococcus (Group D) and Fusobacterium varium ). Teeth and bones were treated with enhanced endodontic surgery as described above. The root canal was completed after 6 weeks with the temporary prosthesis. At that point, the fistula appeared to heal. After 3 weeks, the site appeared to be cured.

A 43 year old woman had an infected maxillary premolar (position 13). Radiographs showed thickening periodontal ligament (“PDL”) in the near extremities, possibly fractured teeth, especially on the mesial plane of the entire root. Enhanced endodontic surgery was performed as described above. The patient realized that she had improved within 24 hours and was asymptomatic at the time of completing the appointment one month later. The PDL significantly decreased in thickness within six months, especially in mesial terms.

The 47 year old female had infected teeth (position 14). The upper left area was swollen and painful in the left upper quadrant. Enhanced endodontic surgery was performed as described above. Pain and swelling disappeared after 1 day of appointment. Within one month, the patient was asymptomatic and closure by filling the duct space was routine.

The 50-year-old woman had pain in the lower left mandible. Investigation revealed that two teeth were the cause. One (position 18) had irreversible pulpitis, and the other (position 19) had a previous root canal site infection and showed periapical radiopacity in its perspective. Both teeth were treated with an enhanced endodontic procedure as described above. The following day, the patient reported no pain after surgery, without analgesic or anti-inflammatory medication. The radiopaque on the perspective of the second tooth disappeared 2 months later when the treatment procedure was completed, and the tooth remained asymptomatic.

A 31 year old woman had chronic cementitis and periodontitis in the teeth (position 19) who had been treated over two months. Radiographs showed bone radiograph at the proximal end. The teeth were re-treated with the enhanced endodontic procedure as described above. Subject reported significant pain relief within a few days. The site was asymptomatic at month 1, ie when the root canal procedure was routinely completed. Radiographs taken 6 weeks later showed that the bone abscess healed.

A 76 year old woman had intermittent pain in her teeth (position 21). The patient's history showed previous heart attacks, cancer and antibiotic allergies. Radiographs showed periapical radiotherapy and calcification of the dental canal. The teeth were re-treated with the enhanced endodontic procedure as described above. The pain was relieved within 1 day. Radiographs of the 1 month period showed the disappearance of bone abscess radiographs.

A 40 year old woman had pain and periapical bone abscess around the teeth (position 18), despite years of intragingal treatment. The teeth were re-treated with the enhanced endodontic procedure as described above. The entire treatment procedure was completed after 6 weeks. At 6 weeks, the radiographs showed a marked improvement and slight resolution of periapical abscess lesions.

An 18-year-old woman had a periapical bone abscess on the myocardium of the tooth (position 19). The extracted dimensions during intragingal treatment were necrotic. The teeth were re-treated with the enhanced endodontic procedure as described above. Final treatment was carried out by conventional methods with beat Perca and sealed with IRM. Radiographs at week 8 showed a slight improvement of the abscess lesions of the apical and anterior roots.

Control subjects

A 58 year old male had teeth with a history of periodontal disease (position 13). Its dimensions were necrotic. Radiographs showed periapical bone abscess lesions. Conventional endodontic treatment was performed with a temporary dressing of Ca (OH) ₂ . Treatment was completed after six weeks. The teeth still had mild symptoms and bone abscesses were still present on the radiographs, perhaps somewhat larger.

A 47-year-old man had a previously prosthetic tooth (position 10) with crown and prostheses, indicating severe resorption of the periapical bone with chronic pain. Conventional endodontic treatment was performed with a temporary dressing of Ca (OH) ₂ . Treatment and beat Perca root canal filling were completed after 6 months. The teeth were still mild and apical bone defects were still present on the radiographs after one year.

A 49-year-old woman had fractured teeth (position 19), which caused pain for a year despite prosthetic prosthesis. The first radiographs showed dentin fractures and periapical bone abscesses. Conventional endodontic treatment was performed with a temporary dressing of Ca (OH) ₂ . Closure and beat Perca root canal filling was completed after 7 weeks. Teeth had mild symptoms and periapical bone defects were still present on radiographs taken 8 months later.

These results, including a comparison of treated and control subjects, suggest that the pharmaceutical formulation is effective at treating intragingal bone abscess and promoting bone regrowth at the treated site.

Example  5: Periodontal  Treatment of lesions and fibroids

The experiments below were performed to determine if the pharmaceutical formulation induces osteomyelitis in residual periodontal ligament tissue. A 38-year-old male was diagnosed with a mildly transmissive radiographic lesion in the tooth (position 32) that was the site of the previous wisdom tooth. History and radiation submissions were consistent with fibroma. Prior to the biopsy, one example of a pharmaceutical formulation with Formula B from Example 3 was injected 1 cc into the lesion using a trocar. Repeated radiographs after 3 weeks showed increased radiation density in the lesion. Post-treatment extraction biopsies confirmed the presence of osteoblasts induced in benign fibrous lesions, irregular trabeculae of woven bone, and osteoblasting. These results suggest that the pharmaceutical formulation is effective at inducing osteogenic metabolism at the site of periodontal fibroma.

Example  6: Treatment of complex fractures

The 25-year-old man suffered a complex fracture of the right humerus in a motorcycle race. On the day of the accident, open surgical correction was performed using multiple screws and plate fixation. Radiographic evidence, despite being fixed, showed wide-ranging, serious injuries in the bone juxtaposition. One month after the operation, the healing response was not clear at all on the radiograph. Subjects received an example of one of the pharmaceutical formulations of Formulation B from Example 3 above four times intravenously over two weeks. Radiographs taken after treatment and 7 weeks after initial surgical repair showed that recovery was facilitated in several gaps on the fracture line. The normal recovery process for this type of injury is estimated to be 4 months. Thus, the results suggest that the pharmaceutical formulation is effective at inducing bone regrowth and promoting recovery of complex fractures.

Example  7: Treatment of horse fractures and joint injuries

I had two horses treated with one of the pharmaceutical formulations.

The first horse, a three-year-old hematoma, was diagnosed as a scapula fracture that had not been dislocated after showing leg swelling and failing to respond to a week of careful action. An example of one of the pharmaceutical formulations of Formulation B from Example 3, above, was injected directly into the fracture line under fluoroscopy control and the legs were fixed with splints. Within six weeks, the fractures healed without callous or malformations, as seen on the radiographs. The horse returned to the race eight weeks after treatment. In normal horses, the time to heal these weight-compression fractures is 5 months for closed fracture correction.

The second horse, a six-year-old horse, showed a bridge, and was diagnosed with an swelling of an infectious etiology in the carpal bone of the right hind limb. Arthroscopic fixation was performed on two carpal bone joints. One carpal bone joint was treated by injecting an example of the pharmaceutical formulation of Formulation B from Example 3 directly into the joint during surgery. One carpal bone joint was used as a control without injection of the pharmaceutical formulation. Radiographs three and six weeks after surgery showed that joint fusion was promoted in joints treated with pharmaceutical formulations, as compared to untreated control joints. At week 6, the cast was removed from the treated joint, which is almost five months faster than conventional horse arthroplasty.

Cited References

The following US patent documents and publications are incorporated herein by reference.

U.S. Patent 4,451,480 to DeVillez

U.S. Patent 4,591,602 to DeVillez

US Patent 4,983,637 to Herman

U.S. Patent 5,086,076 to Herman

U.S. Patent 5,126,376 to Herman

U.S. Patent 5,190,977 to Herman

U.S. Patent 5,190,979 to Herman

U.S. Patent 5,260,342 to Herman

U.S. Patent 5,270,344 to Herman

U.S. Patent 5,364,879 to Herman

Other publications

Claims (26)

Peroxides resulting from the oxidation of alkenes or menthols, including terpineol, citronellol, nerol, linalool, phytol, geraniol, perylyl alcohol, menthol, geranyl geraniol or farnesol with an oxygen-containing oxidant About 0.001 to about 30 weight percent of the species or reaction product based on the pharmaceutical formulation; From about 50 to about 99 weight percent of a permeable solvent, including dimethyl sulfoxide, sterol, lecithin, propylene glycol or methylsulfonylmethane, based on the pharmaceutical formulation; Porphyrin, Rose Bengal, Chlorophyllin, Hemin, Corin, Texaprine, Methylene Blue, Hematoxylin, Eosin, Erythrosine, Lactoflavin, Anthracene Dye, Hypericin, Methylcholanthrene, Neutral Red, Phthalocyanine, Fluores From about 0.1 to about 30 weight percent of a chelated divalent or trivalent metal-containing dye, including sei or peomelanin, on a pharmaceutical formulation basis; And About 0.01 to about 20 weight percent of an aromatic redox compound, including substituted or unsubstituted benzoquinone, naphthoquinone or anthroquinone, based on pharmaceutical formulation A method of stimulating bone regeneration in a patient, comprising administering to the patient an effective amount of a pharmaceutical formulation comprising a. The method of claim 1 wherein the alkene is in liquid form, solution or dispersion. The method of claim 1 wherein the alkene is contained in a fixed oil, ester, fatty acid or ether. The method of claim 1, wherein the oxygen-containing oxidant is selected from the group consisting of singlet oxygen, triplet oxygen, superoxide anion, periodate, hydroxyl radical, hydrogen peroxide, alkyl peroxide, carbamyl peroxide, Benzoyl peroxide, or a method comprising oxygen bound to a transition element. The method of claim 1 wherein the oxygen-containing oxidant comprises ozone. The method of claim 1 wherein the permeable solvent is a liquid, micelle membrane, liposome, emollient or vapor. The method of claim 1 wherein the permeable solvent is dimethylsulfoxide (“DMSO”). The method of claim 1 wherein the dye comprises porphyrin, rose bengal, chlorophylline or mixtures thereof. The method of claim 1 wherein the metal comprises iron. The method of claim 1 wherein the metal comprises copper, manganese, tin, magnesium or strontium. The method of claim 1, further comprising an electron donor. The method of claim 11, wherein the electron donor comprises ascorbic acid or a pharmaceutical salt thereof. Peroxide species or reaction products produced by oxidizing geraniol with a mixture of ozone and oxygen; Dimethyl sulfoxide ("DMSO"); Chelated divalent or trivalent metal-containing dyes, including mixtures of hematoporphyrin and rose bengal or mixtures of hematoporphyrin and chlorophyllin; And Methylnaphthoquinone A method of stimulating bone regeneration in a patient, comprising administering an effective amount of the pharmaceutical formulation to the patient. Peroxides produced by oxidizing alkenes or menthols, including terpineol, citronellol, nerol, linalool, phytol, geraniol, perylyl alcohol, menthol, geranylgeraniol or farnesol with an oxygen-containing oxidant About 0.001 to about 30 weight percent of the species or reaction product based on the pharmaceutical formulation; From about 50 to about 99 weight percent of a permeable solvent, including dimethyl sulfoxide, sterol, lecithin, propylene glycol or methylsulfonylmethane, based on the pharmaceutical formulation; Porphyrin, Rose Bengal, Chlorophyllin, Hemin, Corin, Texaprine, Methylene Blue, Hematoxylin, Eosin, Erythrosine, Lactoflavin, Anthracene Dye, Hypericin, Methylcholanthrene, Neutral Red, Phthalocyanine, Fluores From about 0.1 to about 30 weight percent of a chelated divalent or trivalent metal-containing dye, including cein, eumelanin or peomelanin, on a pharmaceutical formulation basis; And About 0.01 to about 20 weight percent of an aromatic redox compound, including substituted or unsubstituted benzoquinone, naphthoquinone or anthroquinone, based on pharmaceutical formulation A method for increasing osteoblast activity in a patient, comprising administering to the patient an effective amount of a pharmaceutical formulation comprising a. The method of claim 14, wherein the alkene is in liquid form, solution or dispersion. The method of claim 14, wherein the alkenes are included in fixed oils, esters, fatty acids or ethers. 15. The process of claim 14, wherein the oxygen-containing oxidant is singlet oxygen, triplet oxygen, superoxide anion, periodate, hydroxyl radical, hydrogen peroxide, alkyl peroxide, carbamyl peroxide, benzoyl peroxide, or A method comprising oxygen bound to a transition element. The method of claim 14, wherein the oxygen-containing oxidant comprises ozone. The method of claim 14, wherein the permeable solvent is a liquid, micelle membrane, liposome, emollient or vapor. The method of claim 14, wherein the permeable solvent is dimethylsulfoxide (“DMSO”). The method of claim 14, wherein the dye comprises porphyrin, rose bengal, chlorophyllin or mixtures thereof. The method of claim 14, wherein the metal comprises iron. The method of claim 14, wherein the metal comprises copper, manganese, tin, magnesium or strontium. The method of claim 14, further comprising an electron donor. The method of claim 24, wherein the electron donor comprises ascorbic acid or a pharmaceutical salt thereof. Peroxide species or reaction products produced by oxidizing geraniol with a mixture of ozone and oxygen; Dimethyl sulfoxide ("DMSO"); Chelated divalent or trivalent metal-containing dyes, including mixtures of hematoporphyrin and rose bengal or mixtures of hematoporphyrin and chlorophyllin; And Methylnaphthoquinone A method of increasing osteoblast activity in a patient, comprising administering to the patient an effective amount of a pharmaceutical formulation comprising a.