The present invention relates to a method of recycling polymeric waste products into monomers and high value forms of carbon by pyrolytic conversion using an induction coupled RF plasma heated reactor.

Today""s society uses and quickly discards a large volume of an increasingly diverse range of polymeric materials. According to a recent EPA report, in 1997 xe2x80x9cplasticsxe2x80x9d accounted for 9.4% by weight (19.8xc3x97106 tons) of the total municipal solid waste (MSW) generated in the United States (Environmental Protection Agency, xe2x80x9cCharacterization of Municipal Waste: 1997 update, EPA Report 530-R-98-007, Franklin Assoc. Ltd., Prairie Village, Kans., May (1998)). Of this, only 5.3% (1.1xc3x97106 tons) is recycled into low-grade applications such as synthetic lumber for park benches. The rest is either incinerated or landfilled. However, incineration is generally associated with the generation of NOx and other hazardous emissions while the cost of landfilling in many areas now exceeds $100/ton due to tighter federal and state regulations and decreases in the available land. Further, polymers from petrochemical sources have a very slow rate of biodegradation. Thus, the need to recycle MSW plastics is becoming increasingly important.
Most conventional chemical methods cannot deal with MSW due to the diversity of its composition, which ranges from single thermoplastics to complex thermosets and composites.
Thermal plasma processing of mixed industrial waste streams containing metallic, inorganic or organic components have been reported (Montgomery, R. W. Proc. 11th Intl. Symp. Plasma Chem. (ISPC-11), IUPAC, Loughborough, UK, August 22-27, Vol. 2, 526-530 (1993); Taylor, P. R. and Pirzada, S. A. Adv. Performance Mater. 1, 35-50 (1994); and Hoffelner, W. and Funfschilling, M. R. Proc. Workshop on Ind. Appl. of Plasma Chem., 12th Intl Symp. Plasma Chem. (ISPC-12), IUPAC, Minneapolis, Minn., August 25-26, Vol. B. Thermal Plastic Applications, 3-7 (1995)). These processes generate both solid (metal +slag) and gaseous product streams. Gaseous product streams are typically in the form of xe2x80x9csyn-gasxe2x80x9d which is a mixture of CO and H2 usable as a fuel or as a precursor for organic syntheses. The thermal plasma gasification of particulate coal and processing of methane gas have also been reported for the synthesis of acetylene (Gannon, R. E. Ind. Eng. Chem. Prod. Res. Develop. 9(3), 343-347 (1970)). Further, thermal plasma destruction of toxic chemicals such as PCBs and CCl4 have been disclosed (Smith et al. Proc. 3rd Euro. Cong. Thermal Plasma Process (TPP-3), VDI, Aachen, Germany, September 19-21, 667-674 (1994); Tock, R. W. and Ethington, D. Chem. Eng. Comm. 71, 177-187 (1988); Han et al. J. Mater. Synth. Process 1(1), 25-32 (1993); Lachmann et al. Proc. 3rd Euro. Cong. Thermal Plasma Process (TPP-3), VDI, Aachen, Germany, September 19-21, 591-597 (1994); Mosse, A. and Kusnetov, G. Proc. 3rd Euro Cong. Thermal Plasma Process (TPP-3), VDI, Aachen, Germany, September 19-21, 651-657 (1994); Breitbarch et al. Plasma Chem. Plasma Proc. 17(1), 39-57 (1997); and Sekiguchi et al. Plasma Chem. Plasma Process 13(3), 463-478 (1993)). In addition, thermal plasma vitrification of high and low level nuclear waste has been reported as a means of reducing the volume of the waste and of encapsulating it in a non-leachable matrix prior to burial/storage (Hoffelner, W. and Funfschilling, M. R. Proc.
Workshop on Ind. Appl. of Plasma Chem., 12th Intl Symp. Plasma Chem. (ISPC-12), IUPAC, Minneapolis, Minn., August 25-26, Vol. B. Thermal Plastic Applications, 3-7 (1995)); Girold et al. Thermal Plasma for Hazardous Waste Treatment, Benocci et al. eds. World Scientific, Singapore, 160-168 (1996); Munz, R. J and Chen, G. Q. J. Nucl. Mater. 161, 140-147 (1989); and Hoffelner et al. Thermal Plasmas for Hazardous Waste Treatment, Benocci et al. eds. World Scientific, Singapore, 126-145 (1996)). The primary advantage of thermal plasma processes over other technologies is their ability to process solid, liquid and gaseous materials, rapid thermal response and quenching, and low off-gas emission compared with combustion-driven incinerators.
Thermal plasmas can be generated by direct current (DC) or alternating current (AC) arc/plasma systems utilizing either consumable or non-consumable electrodes or by electrodeless radio frequency induction-coupled plasma systems (ICP) (Fauchias et al. in Thermal Plasma for Hazardous Waste Treatment, Benocci et al. eds, World Scientific, Singapore, 1-38 (1996)). The majority of thermal plasma/waste processes developed have used DC plasma generators.
However, DC thermal plasma systems for waste treatment are limited in applicability for several reasons. First, DC plasma systems generally suffer from electrode erosion and product contamination from electrode erosion. Second, scale-up is limited by electrode erosion, and until fairly recently by power supply technology. Finally, the control of the process chemistry is difficult in DC systems since generally only inert, nonoxidizing plasma forming gases can be used. Thus, the importance of ICP systems is beginning to be recognized (Smith et al. Proc. 3rd Euro Cong. Thermal Plasma Process (TPP-3), VDI, Aachen, Germany, September 19-21, 667-674 (1994); Huhn et al. Pract. Period Hazardous Toxic and Radioactive Waste Management , 107-117, July (1997); Boulos Proc. Workshop on Ind. Appl. of Plasma Chem., 12th Intl Symp. on Plasma Chem. (ISPC-12), IUPAC, Minneapolis, Minn., August 25-26, Vol. B, Thermal Plasma Applications, 89-95 (1995); Boulos, M.I. IEEE Trans. Plasma Sci. 19(6), 1078-1089 (1991)).
The focus of thermal plasma technologies has been on destruction of hazardous chemical wastes such as chlorinated and fluorinated hydrocarbons, the extraction of metals from industrial waste, volume reduction/vitrification of nuclear waste and the destruction of military waste.
In the present invention, a method is provided for recovering monomers and high value carbons from polymeric wastes using a thermal plasma treatment process.
An object of the present invention is to provide a method of recovering monomers and high value carbons such as carbon nanotubes and Fullerenes from polymeric wastes using an RF induction-coupled thermal plasma heated reactor operating close to atmospheric pressure. In this method, powders of polymeric wastes are injected into an induction coupled RF plasma heated reactor wherein the polymeric waste powders are pyrolytically converted to monomers and high value forms of carbons. These monomers and high value forms of carbons are then recovered from the reactor.
A significant, valuable percentage of today""s municipal solid waste stream is polymeric materials. In fact, typical polymeric municipal solid waste comprises approximately 25.36% polyethylene (LDPE/LLDPE), 20.85% polyethylene (HDPE), 13.06% polypropylene (PP), 10.07% polystyrene, 8.61% polyethylene terepthalate (PET), 6.23% polyvinyl chloride (PVC) and 15.84% other materials. This polymeric waste is generally incinerated, landfilled or recycled via downgraded usage as economic recycling technologies have not been available.
In the present invention, however, an induction coupled RF plasma heated reactor is used for the pyrolytic conversion of olymeric materials to monomers and high value carbon forms including, but not limited to Fullerenes, including carbon nanotubes. It has now been found that a powder of waste polymers can be converted to monomers by injecting the powder axially into the center of a plasma, preferably an argon plasma. Specifically, the methodology of the present invention converts powdered polymeric materials into high levels of ethylene and/or propylene. Analysis of the gaseous product stream of the reactor demonstrated that using the method of the present invention, high levels of ethylene and propylene were obtained, together with lesser amounts of hydrocarbons including, but not limited to, methane, acetylene and 1,3-butadiene. Typically, approximately 50% conversion of the solid feedstock for polyethylene and 75% conversion of the solid feedstock for polypropylene was obtained.
Further, some solid carbonaceous residues were also produced and collected in the reactor during operation. Analysis of these residues showed the presence of carbon nanotubes, one example of the family of novel, high value, carbon forms ,Fullerenes.
Fullerenes are commonly produced via thermal plasma synthesis using an electric arc discharge between carbon electrodes in a helium atmosphere. Glow and arc plasmas, generally of the direct current (DC) type, have also been described for the synthesis of Fullerenes, including nanotubes (Wilson, E. CandEN, page 11-12, Nov. 9, 1998).
Nanotubes were first discovered less than a decade ago. They are typically synthesized by various vapor decomposition processes, including the catalytic decomposition of acetylene in nitrogen at temperatures above 700xc2x0 C., and the plasma-enhanced chemical vapor deposition (PE-CVD) from acetylene in ammonia. The production of single-walled carbon nanotubes by a chemical vapor deposition (CVD) process has been disclosed as a means for incorporating nanotubes into conventional microstructures (Baum, R. CandEN, page 11, Nov. 9, 1998). CVD techniques typically operate at low pressures, thus requiring complex vacuum systems. These systems are also limited in both throughput and scale-up.
In the present invention, however, high value novel forms of carbon are produced via atmospheric pressure thermal plasma synthesis from polymeric waste products. Thus, the method of the present invention provides an environmentally friendly alternative to polymeric waste recycling as compared to landfilling and/or incineration and a useful method for production of novel, high value carbon products, including, but not limited to, Fullerenes.
In this method, polymeric waste products are first made into powders. In a preferred embodiment, the powders comprise free flowing particles. Thus, particles having a spherical morphology are preferred. Particle size is dependent upon the apparatus being used. In experiments described herein, powders comprising particles approximately 5 to 90 xcexcm were used. After grinding or chopping of the polymeric waste products, it is preferred that resulting powders be seived or screened prior to introduction into the reactor so that particles in the powder are within a selected size distribution. The powders are then introduced into an induction coupled RF plasma heated reactor wherein the polymeric wastes are pyrolytically converted to monomers and high value forms of carbon. These monomers and high value forms of carbon are then recovered from the reactor.
A series of experiments were conducted to understand the reaction mechanisms involved in the plasma conversion of waste polymers back into monomers and other useful compounds including carbon nanotubes via the method of the present invention. The key process parameters believed to be most important to the yield of monomers are listed in the following Table 1, along with optimal values determined from experiments with polyethylene (PE) and polypropylene (PP).
A two level factorial experimental matrix was designed using a statistical software package (DESIGN-EASE(trademark)) to determine the principal parameters affecting the yield of products. Only the first 6 parameters in Table 1 were considered in this analysis. Eight experiments were required to study the effects of all 6 parameters and 8 more were needed to separate the main effects from interactions.
From the initial set of experiments, as analyzed using DESIGN-EASE(trademark), the standardized effect of each parameter plus their interactions on the amount of ethylene and propylene produced was obtained. A plot of standardized effect versus normal probability showed a straight line through most of the points. For proper use of a normal probability plot, the effects must have a common error variance. Missing data or altered independent factors can cause the variance associated with the estimated effects to differ. The effects must therefore be standardized to correct for this (Box, G.E.P., Hunter, W. G. and Hunter, J. S. Statistics for Experimenters: An Introduction to Design, Data Analysis and Model Building, John Wiley and Sons, NY (1978)). Standardized effects were computed using DESIGN-EASE(trademark) in conjunction with the following equation, wherein A represent the first variable considered:
Standardized Effectj=(Coefficienti) (2) (Std. ErrorA/Std. Errori)
Those factors which were significantly off the line were the variables of statistical importance. These were: plasma plate power input, central gas flow rate, probe gas flow rate, power feed rate, and interaction between the plasma plate power and quench gas flow rate. Even though the plot indicated that the latter variable was not very significant, it was chosen in order to study the interaction. System constraints limited the range of flow for the probe gas, so the optimum value determined during preliminary runs was used and kept constant in subsequent experiments.
The influence of plasma power plate input over the range from 10 kVA to 20 kVA on the production of ethylene was determined. There was a small reduction in the ethylene concentration at the higher power level. Because power level also interacted with other variables, no generalization can be made for this results. However, by plotting the data, it was deduced that at the higher plasma plate power level more quench gas was required to produce ethylene. This indicated that the higher average temperature of the plasma region, when less quench gas was used, favored the production of other species such as propylene. Similar analysis of other parameters led to the choices for optimal values for the variables set forth in Table 1.
The gaseous products were mostly mixtures of propylene and ethylene, together with small fractions of other hydrocarbons, such as methane, acetylene and 1,3-butadiene. Product distribution for the optimum case conditions is listed in Table 2.
Although ethylene concentration was the parameter used for optimizing the variables in polyethylene recycling, propylene was also present in the product gases by a factor of 2.4 times greater than for ethylene. In polypropylene recycling, propylene was present in the product gases by a factor of 30.3 times greater than for ethylene. For polyethylene, other hydrocarbons were present to a lesser extent (10% to 25% of ethylene) For polypropylene, other hydrocarbons were present at similar concentrations to ethylene. The total concentration of hydrocarbons listed in Table 2 on an argon free basis for polyethylene was 90% which indicated that there were other products such as hydrocarbons and hydrogen not detected by the gas chromatography analysis.
Variations in concentration of ethylene and propylene with plasma plate power were also determined. For polyethylene recycling, it was found that ethylene concentration decreased from 22% to 10% when the power was increased from 10 kVA to 20 KVA; propylene concentration increased over the same power range from 53% to 78%. Methane concentration decreased at a slower rate. For polypropylene recycling, increased power had little effect on concentrations of monomers recovered.
Variations in the products as the central gas flow rate of argon increased from 15 to 40 slm were also determined for polyethylene recycling. It was found that ethylene concentration increased by 37.4% over the flow range studied. An increased scatter in the data, believed to be caused by turbulence in the plasma zone, was observed. This is indicative of a shorter residence time in the plasma regions and a greater variation in the effective temperature. A small increase in methane concentration was also observed. Acetylene increased from about 0.8% at 15 slm to 5.4% at 40 slm. The 1,3-butadiene increased from 1.5% at the lower flow to 2.5% at the higher flow. The decrease in the propylene/ethylene ratio also indicated a lower average plasma temperature. Propylene concentration was also observed to decrease from 71.2% to 51.7% over the same range of variation in central gas flow rate.
The influence of quench gas flow rate was also determined. In polyethylene recycling, as the quench flow rate varied from zero to 155 slm, the ethylene concentration peaked at 23.2% at a quench flow rate of 29 slm. Again there was greater scatter in the data at the higher quench flow rates, indicating that increased turbulence led to variations in the average temperature. Similar trends were observed in polypropylene recycling.
The ratio of quench gas flow rate to the sum of sheath, central and probe gas flow rates was a maximum of 1.75. The quench gas flow rate thus did not affect the structure of the plasma to a significant extent. It only affected the tail flame of the plasma zone. Ethylene concentration decreased with the increase in the quench gas flow rate. This indicated that the ethylene was mainly produced in the plasma tail flame region, which was most affected by the quench gas. The maxima in the ethylene concentration curve at low quench gas flow rates could have been due to quenching of any secondary reactions occurring in the hot gases exiting the plasma zone.
To determine whether the quench velocity rather than the total gas flow was the controlling variable, quench nozzles of different orifice diameters were used. As the velocity of the quench gas was increased by decreasing the nozzle orifice diameter, the flow rate at which maximum ethylene concentration occurred decreased.
PE particle feed rate was varied from 1.5 to 5.0 grams/minute. The ethylene concentration approximately doubled from 11.97% to 22.24% as the feed rate increased from minimum to maximum. Concomitant to the ethylene production, the solids recovered from the reactor increased from 15% of those fed at the 1.5 gram/minute level to 50% at the 5 gram/minute level of feed.
Transmission electron microscopy (TEM) analysis of the solid material recovered from recycling of both polyethylene and polypropylene indicated a range of carbon structures, from highly oriented crystalline structures through turbostratic to completely amorphous. Carbon nanotubes were also detected, thus indicating that, not only is this process useful in the recycling of polymeric or hydrocarbon feedstocks but also in the production of high value carbon materials from these feedstocks.

Thermal plasma process for recovering monomers and high value carbons from polymeric materials


A hernia belt which is provided in two parts, comprising an abdominal belt portion, having a pair of leg straps which extend downwardly for passing between the legs of a wearer, and a separate back belt portion, which extends around the back of the wearer. Virtually the entire face of the exterior of the abdominal belt portion is formed of hook or loop material, which engages with mating loop/hook surfaces on the ends of the back belt portion. The combination of the abdominal belt portion having virtually its entire surface covered with hook/loop material, and a back belt portion with hook/loop material at its ends, makes it possible to provide virtually universal or xe2x80x9cfits allxe2x80x9d hernia support.

The present invention is directed to a hernia belt or truss useful in supporting abdominal hernias.
Hernia belts have been well known for many years and have been proposed and used in many different forms. Examples of some of these differing devices are reflected in the patent literature.
U.S. Pat. No. 4,059,103, issued Nov. 22, 1977, to Glaser, describes a one-piece bi-sexual garment. A narrow neck section of the support passes through the crotch of the wearer and anchors the bottom part, while a first belt like elastic band connects and supports the upper parts of the front and rear sections, and is secured to both. A second belt-like elastic band encircles the hips of the wearer below the first elastic band, and is fastened only to the rear portion of the support. The elastic bands extend over the hips to provide up-lift.
U.S. Pat. No. 4,671,264, issued Jun. 9, 1987, to Frangi, describes an underpant brief and hernial truss. An elastic belt is disposed at the circumferential upper edge of the brief, and a pair of at least partly elastic bands has upper ends which are affixed to the elastic belt and pass laterally of the crotch of the pant, adjacent the leg holes of the brief. These bands include an inelastic segment on the front of the underpant extending diagonally across a hernia affectable region of the wearer. Pockets are included over the hernia affectable region for receiving a retaining pad.
U.S. Pat. No. 4,416,272, issued Nov. 22, 1983 to Nelkin, describes another combination of underpant and hernial truss. This patent describes a brief having a front, rear and crotch portions with spaced leg holes. A truss pad is attached to the front portion and protrudes inwardly to the brief to engage and support a herniated abdominal area. An adjustable length belt encircles the brief and is connected to the front portion adjacent the pad for directing hernia retaining force thereon. The belt is free from securement to the rear portions of the brief for selective movement upwardly and downwardly to adjust to the wearer""s comfort.
A clean copy of amended page 2 is attached.
U.S. Pat. No. 4,351,325, issued Sep. 28, 1982, describes a wide roll-on belt to which the pressure pad for bearing on the area of the hernia is indirectly attached by an arrangement including a rigid stay member. A strap extends from a lower edge of the pad between the wearer""s legs to keep the pad pressed against the body by reaction with the rigid stay, even when movement tends to displace the adjacent part of the belt away therefrom, as when sitting or stooping.
U.S. Pat. No. 4,787,379, issued Nov. 29, 1988, to Yeh, describes a hernia truss comprising a belt which encloses a flexible and bendable spring metal band. Attached to one end of this belt is a soft pad supported upon a round metal plate. The pad is intended to overlie the hernial region when the ends of the belt are fastened together.
U.S. Pat. No. 3,754,549, issued Aug. 28, 1973, to Nelkin, describes an essentially one piece truss belt which is adapted for adjustment in circumference. The adjustment entails the belt being open at the back and having a rather complex fold and strap arrangement for determining the belt circumference.
While the foregoing prior devices purport to serve their intended purposes, for the most part, their designs are such as to produce a significant degree of complexity of adjustment and/and or uncomfortableness for the wearer.
The invention relates to a hernia belt which is provided in two basic parts which together provide a maximum of adjustability, adaptability, and comfortableness. The two piece hernia belt comprises a soft abdominal belt portion, which is adapted to extend around the abdomen and carry one or two removable hernia pads. Attached to this abdominal portion of the belt are a pair of spaced leg straps which extend downwardly for passing between the legs of a wearer. The abdominal portion of the belt is held in place on a wearer by adjustable attachment to a separate back belt portion, which extends around the back of the wearer.
According to the invention, virtually the entire face of the exterior of the abdominal belt portion is formed of hook or loop material, preferably loop material. This hook/loop surface engages with mating loop/hook surfaces on the ends of the back belt portion. By this combination of an abdominal belt portion having virtually its entire surface covered with hook/loop material, and a sufficiently long back belt portion with hook/loop material at its ends, it is possible to provide virtually universal or xe2x80x9cfits allxe2x80x9d hernia support.
At the same time, the leg straps which depend from the abdominal portion of the belt are adapted to be adjustably secured to the back belt portion through hook/loop attachment. This adjustable leg strap arrangement interacts with the adjustable waist size in providing the virtually universal size hernia belt. The abdominal belt portion carries one or more pockets for receiving one or more pads (right or left, or right and left), adapted to provide the desired hernial support.
It is a primary of object of the present invention to provide an improved hernia belt, which eliminates or alleviates the shortcomings of the foregoing exemplar hernia belts described in the patent literature.
It is another object of the invention to provide such an improved hernia belt which provides easy adjustment, is comfortable to the wearer, and which provides a fit for virtually all waist sizes.


The invention relates to novel microemulsions comprising a non-polar lipid, at least one polar solvent, a surfactant, and a polar lipid. A microemulsion of these ingredients provides an environment that substantially encloses airborne particles, and it can be used for entrapping such particles. The inventive microemulsions are especially adapted for the prevention of symptoms in mammals, which are indirectly or directly caused by airborne particles. The invention further provides compositions comprising such microemulsions and methods of treatment comprising administering the same.

Airway diseases caused by exposure to airborne allergens are major health problems to affluent societies. For example, the prevalence of allergic rhinitis (seasonal, perennial, and occupational) is 20% or more, and increasing. Accordingly, the American Asthma and Allergy Foundation recently reported that about 17 million annual visits to office-based physicians were attributed to allergic rhinitis. Also, they reported that 35 million Americans were allergic to pollen allergens. Besides the morbidity and socioeconomic consequences that these allergies carry themselves, they have been identified as risk factors for the development of asthma. Indeed, one way of preventing the development of bronchial asthma may be to treat the allergic rhinitis condition.
Upper respiratory tract infections (caused by viruses and bacteria) are also major health problems. For example, the common cold is a very prevalent disease: The number of common cold episodes has been estimated to be 5 per year in the 0-4 year old age group, dropping to 3 episodes per year in the 5-19 year old group.
These infections have, notably in the case of the common cold, been identified as a major cause of exacerbations of asthma and "chronic obstructive pulmonary disease".
"Non-allergic, non-infectious (vasomotor) rhinitis" is a less prevalent condition (compared with allergic rhinitis and upper respiratory tract infections). Nevertheless, it is a common differential diagnosis of patients having nasal symptoms.
Pollens and allergens themselves, as well as viruses, bacteria, and other exogenous factors, occur as airborne particles. Pollens typically have diameters between 15-60 μm. Virus particles, on the other hand, are typically 0.1-0.5 μm in diameter, while the size of bacteria and other prokaryotes ranges widely from 0.1 to about 5 μm. Cigarette smoke, printing ink, and occupational factors, which may produce "non-allergic, non-infectious (vasomotor) rhinitis", may also be viewed as particles with a wide size range. In order to produce disease-processes and symptoms associated with allergic rhinitis, upper respiratory tract infections, and "non-allergic, non-infectious (vasomotor) rhinitis" the disease-producing particles must come in contact with the mucosal surface.
Mechanisms leading to allergic rhinitis comprise the contact of inhaled pollens/allergens with the nasal mucosal lining and the interaction with immunocompetent and inflammatory cells residing in the nasal mucosa. Specific antibodies (immunglobulins) are located at the surface of some of these cells. The allergen cross-bridges with receptors on the cell surface, which results in an activation of these cells and in the release of inflammatory mediators. These mediators, in turn, act on certain cells and structures in the mucosa (e.g., glands, blood vessels, nerve endings) to produce inflammation and the symptoms of allergic rhinitis, i.e., nasal itching/sneezing, secretions, and blockage.
Upper respiratory tract infections are caused by viruses and bacteria interacting with the mucosa of the upper airways. The most common cause may be rhinoviruses, which bind to specific mucosal receptors, an inflammatory response being produced. This process, in turn, produces symptoms of common cold (sneezing, watery secretions, reduced nasal patency and catarrhal symptoms with accompanying fever and headache)
"Non-allergic, non-infectious (vasomotor) rhinitis" is sometimes caused by airborne particles other than allergens and infectious agents. The pathophysiological processes give rise to a symptomatology that is very similar to that of allergic rhinitis. However, it is less clearly understood.
The best treatment for type-I allergic diseases, such as allergic rhinitis, would be to avoid the allergen. However, this strategy is more or less impossible, particularly for airborn allergens including those carried by pollens. In addition, this line of reasoning applies for infectious agents and particles causing "non-allergic, non-infectious (vasomotor) rhinitis".
Pharmacological treatments for allergic rhinitis typically aim to mediate the inflammatory pathways outlined above. Commonly use current treatments include topical, oral and systemical gluco-corticosteroids, topical and oral antihistamines, topical chromoglycates, oral leukotriene-antagonists, topical and oral decongestants, topical and oral anticholinergics, and immunotherapy, i.e., repeated exposure to relevant allergens in order to produce a state of increased tolerability. However, there is no single pharmaceutical preparation/treatment available, which treats or reduces all symptoms of allergic rhinitis and such inflammation modulating treatments are not without potential side effects. The most successful treatment may be topical corticosteroids, but their use is associated with a potential risk of local and systemic adverse effects. Antihistaminic preparations are commonly used, but their efficacy is limited. Local decongestants, though used, are not recommended by physicians skilled in the art.
There is currently no established pharmacological treatment for upper respiratory tract infections, except for antibiotics and other chemotherapeutics targeting bacteria, antiviral compounds targeting viruses, specifically influenza virus, as well as topical and oral decongestants.
Antibiotics may be used to treat certain upper respiratory tract infections caused by bacteria. However, no treatment is available for the common cold (caused by viral infections) except for oral and nasal decongestants (alpha-agonists). The neuraminidase inhibitor zanamivir has recently been introduced for the treatment of Influenza A and B, but its efficacy is limited.
Treatments for "non-allergic, non-infectious (vaso-motor) rhinitis" include topical glucocorticosteroids, topical anticholinergics, and topical capsaicin. Common for these treatments, however, is their limited efficacy.
An attempt to overcome nasal mucosal exposure of airborne particles, including pollens, is shown in U.S. Pat. No. 6,109,262, a special adhesive nasal filter being used, which is physically placed within the nasal passages and collects airborne particles. The method and filter disclosed is said to have therapeutic, protective or prophylactic uses.
Inter. Arch. Allergy Immunol. 121:85, 2000, teaches the usefulness of regularly applying a mineral oil to the nasal mucosa of patients with seasonal allergic rhinitis in order to reduce allergic symptoms. It is suggested in this document that fatty acids might be helpful in the prevention of allergic rhinitis. Topical application of fats, such as certain triglycerides and oils to the nasal mucosa, has also been marketed for treating dryness of the nasal mucosa (Nozoil®, Pharmacure Health Care AB, Sweden)
In DE 20016125 a nasal ointment for prophylaxis of inhalation-allergic reactions, especially hay fever, is shown. The ointment comprises a mixture of saturated hydrocarbons and tannin(s), a mechanical barrier film being formed that prevents allergens from entering the nasal mucosa. Likewise, JP 07258070 depicts a nasal rinsing agent for treating allergic rhinitis. The nose cleaning agent, which consists of an oil/water type of emulsion of pH 3.5-5.5, forms an oil film after spraying into a nasal cavity. As in DE 20016125, the oil is spread on the nasal mucosa and a non-homogenous film is formed.
The attachment of most rhinovirus subtypes to cells depends on a cellular receptor, the intercellular adhesion molecule 1 (ICAM-1). JAMA 1999, May 19; 281(19):1797-804, teaches that recombinant soluble ICAM-1, inhaled as a solution or as a powder, reduces the severity of experimental rhinovirus common colds.
Although the above methods attempt to extract airbourne particles prior to reaching the mucosal surfaces, or to form a barrier between the air and the surface, none are believed particularly effective and none are generally indicated as effective against a range of inflammatory conditions. In particular, the films of oil generated are not found to provide effective protection.
Accordingly, there is an obvious need for new and more efficacious treatments also in this topical area. Furthermore, like for allergic rhinits, there is a need for a way of preventing or reducing, in a preventive way, the above-mentioned symptoms associated with upper respiratory tract infections, such as common colds. It would be most desirable if a single composition were effective against a range of such conditions.
It would thus be evidently advantageous to provide a preparation capable of capturing airborne particles, whereby to prevent the above-mentioned problems.
It would be a further advantage to provide preparations that are able to capture airborne particles and retard or inhibit their interactions with exterior mucosal membranes.
It would be still further advantageous to provide preparations that follow the mucus when swallowed or sneezed out (i.e. are eliminated from the body therewith).
These objectives are accomplished by a microemulsion comprising a surfactant, at least one polar solvent, a nonpolar lipid, as well as a polar lipid.
We have now, surprisingly, found that airborne particles, exemplified by virus, bacteria, pollen, allergens, dust, mould, fungus spores, animal dander, and other solid aerosol particles, can be trapped by certain micro-emulsions and thereby their tendency to cause respiratory tract conditions reduced. No similar microemulsion has hitherto been disclosed.
In a first aspect, the present invention thus provides a microemulsion comprising a non-polar lipid, at least one polar solvent, and a surfactant, characterized in that it further comprises a polar lipid.
In a further aspect, the present invention provides a microemulsion suitable for entrapping airborne particles, characterised in that it comprises or consists of a non-polar lipid, at least one polar solvent, a surfactant, a polar lipid and optionally inert carriers and/or excipients, an environment being provided that substantially encloses said particles. In one preferable embodiment, these are the sole ingredients of the microemulsion.
The microemulsions of the present invention are highly useful, both in themselves and in combination with pharmaceutical agents, when formulated as compositions suitable for application to a mammalian subject. In another aspect, the invention thus provides a composition suitable for administration to peripheral membrane linings of, for example, the nose, the eyes, the ears, the pharynx, and/or the larynx of a mammal, characterized in that it comprises a pharmaceutically effective amount of a microemulsion, especially a microemulsion of the invention. Such compositions may, in one embodiment also comprise a drug agent.
In a yet further aspect, the present invention provides a method for preventing airway diseases in a subject, caused directly or indirectly by airbourne particles, said method comprising contacting at least one surface of said subject with a microemulsion or composition of the invention and in a still further aspect the present invention provides a method of preventing airborne particles reaching exterior mucosal membranes of a mammal, said method comprising the step of administering to said exterior mucosal membranes of said mammal a prophylactically effective amount of a microemulsion or composition of the invention.
In a yet still further aspect, the invention provides a microemulsion of the invention for use in therapy and in a still further aspect the invention provides the use of a microemulsion or composition of the invention in the manufacture of a medicament for the treatment or prevention of a disease caused directly or indirectly by airbourne particles, including airway diseases caused by airborne pollen, bacteria and/or viruses.
Without being bound by theory, it is believed that the invention utilizes the inherent properties of an airborne particle. Typically, such a particle can have a hydrophobic core and is at the same time electrostatically charged. Thereby the particle is conferred hydrophilic properties. However, the charge will immediately be lost when entering the nose and contacting a mucous membrane, which results in that the hydrophobic properties will dominate. Thus, when airborne, such particles (pollens etc.) have a hydrophilic surface which must be substantially captured within a hydrophilic environment.
The inventors now believe that oils will not effectively prevent symptoms caused by air-borne particles because such particles are not efficiently wetted by the oils. The oils cannot discharge the surface static charge on the particles and thus cannot wet or encapsulate them.
Similarly, although aqueous systems may discharge the surface of particles, there is then exposed a hydrophobic surface which is not efficiently encapsulated by palor media such as water. It is thus important to note that the nature of the present micro-emulsions is substantially such that they do not "break" to produce an oil film upon application but remain as micro-emulsions.
Microemulsions typically look like clear solutions, but are thermodynamically stable dispersions of one liquid phase into another. The non-polar and polar (e.g. oil and water) domains in the micro-emulsion exhibit a disordered and intricate microstructure. The extremely low interfacial tension of the oil/water interface, the small droplet diameters of approximately 100 nm or less as well as water domains of a suitable size, are advantageous properties for capturing airborne particles.
Average (mode) droplet sizes (diameters) in the micro-emulsions of the invention will generally be 5 to 200 nm, particularly 10 to 150 nm and more preferably 20 to 100 nm. It is preferable, in order to preserve the micro-emulsion nature of the composition, that no detectable proportion of droplets (for example less than 0.1%) exceed 500 nm, preferably no more than this will exceed 350 nm and most preferably no more than 0.1% will exceed 250 nm. Droplet sizes may be measured by techniques well known to those of skill in the art. In particular, laser light scattering/diffraction methods are standard. Methods involving techniques such as Rayleigh Scattering or cryo-TEM may, however, be necessary to examine particle sizes below around 300 nm.
It is the inventor's non-binding theory that airborne particles can be trapped by the specific self-assembled hydrophilic/hydrophobic structure provided by means of the invention. The micro-emulsions of the invention are able to provide an environment, which substantially encloses (surrounds) the particles.
In addition, the inventive microemulsion need not contain any active agent, such as a drug or the like. In this embodiment, no side-effects originating from such agents are thus obtained.
Furthermore, a sprayable microemulsion can be provided, which contains large amounts of a polar solvent in order to reduce the viscosity. In addition, the microemulsions of the invention are able to top spread homogenously on the mucosa and do not only function as a protective barrier but also as a capturing agent for airborne particles, which follows the mucus when swallowed or sneezed out. In this way particles are efficiently captured, prevented from interaction with the mucosal surfaces and then removed from the area of interaction.
The microemulsions according to the invention are effective for the protection of mucosal membranes that line all body cavities which are open to the exterior. Thus, peripheral membrane linings of the nose, the eyes, the ears, the pharynx, and the larynx can be protected. However, it is preferred to be used for capturing particles in the nasal cavity, the pharynx, and/or the larynx.
A microemulsion can be defined as a thermodynamically stable, transparent dispersion of two immiscible liquids, stabilized by an interfacial film of surfactants. In this connection a microemulsion is a clear thermodynamically stable dispersion of two immiscible liquids containing appropriate amounts of surfactants and co-surfactants.
A microemulsion generally comprises at least one continuous phase and a further phase which typically comprises discontinuous, dispersed "droplets". It will be clear to one of skill in the art that, depending upon the type and proportion of lipids chosen for a particular emulsion, these droplets may be disordered, "liquid" droplets, and/or may have a regular or irregular internal structure, such as that of a cubic or hexagonal liquid crystalline, lamellar, or "sponge" phase. All of such droplets will evidently be included in the scope of the microemulsions described herein. In one preferred embodiment, the droplets comprise liquid phase non-polar lipid.
The meaning of surfactant as used in the description and in the claims follows the current usage well known to those skilled in the art. A good surfactant should have a low solubility in both the bulk and dispersed phases of the microemals ion.
The surfactant is usually an organic polymer with a long chain hydrophobic tail and a small hydrophilic head group, and it can be anionic, nonionic, cationic or ampheoteric. Nonionic surfactants are preferred since they are less harmful to humans than ionic surfactants.
Suitable nonionic surfactants are polyoxyethylenated alkylphenols (mostly p-octyl-, p-nonyl-, p-dodecyl-, di-nonylphenols), polyoxyethylenated straight chain alcohols, polyoxyethylenated polyoxypropylene glycols, polyoxyethylenated mercaptans, long chain carboxylic acid esters (glyceryl and polyglyceryl esters of natural fatty acids, propylene glycol, sorbitol, and polyoxyethylenated sorbitol esters, polyoxyethylene glycol esters) and alkanolamines (diethanolamine-, isopropanolamine-fatty acid condensates) The edible surfactants are mainly the esters based on glycerol, sorbitol, and propylene glycol.
Preferably, the surfactant is an amphiphilic compound such as a polysorbate, a poloxamer, or a fatty acid poly-oxyethylene. It is most preferred that the amphiphilic compound is polysorbate 80.
Suitable ionic surfactants are succinic, citric, and diacetyl tartaric acid esters. Lecithin is the preferred surfactant containing a positive charge.
The required amount of surfactant is dependent on the other components used in the inventive microemulsion. However, the surfactant concentration should always exceed its critical micellar concentration (CMC).
The hydrophilic/hydrophobic balance, i.e. the molar ratio of hydrophilic to hydrophobic moieties, has a temperature maximum in the mutual solubility of the surfactant in water and oil. This maximum is adapted to encompass a temperature interval, within which the inventive microemulsion is intended to be used, for example around room and body temperatures. Preferably, the hydrophilic-hydrophobic balance of the surfactant, should not exceed 7. Thus, the surfactant comprises 0.1 to 20% (wt/wt), preferably 1 to 10% (wt/wt) of the microemulsion.
The surface tension between the two liquid layers of the microemulsion can be further lowered to approach zero by the addition of at least one polar solvent. Thus, the polar solvent may act as a cosurfactant in the inventive microemulsion and is preferably a small molecule that improves the stability of the microemulsion system. Increased amounts of polar solvent also reduces the viscosity, a better micro-viscosity being obtained in the microemulsion droplets.
Typically, such polar solvents are short chain alcohols, e.g. ethanol to butanol, glycols, such as propylene glycol, or medium chain alcohols, amines, or acids. Preferred polar solvents are propylene glycols and/or polyethylene glycols and/or a saline solution.
Water and buffered aqueous media can also be used for solubilizing one phase into the other in the microemulsion system, which contain a variety of salts and buffers. Preferably, the salts are alkali and alkaline earth halides, e.g. sodium chloride, potassium chloride, or sodium sulphate. Various buffers may be used, such as citrate, phosphate, HEPES, Tris, or the like to the extent that such buffers are physiologically acceptable for its purpose.
Evidently, the polar solvent may be included in the microemulsion at the time of application, and this forms a preferred aspect of the invention. In an alternative aspect, however, the polar liquid may be incorporated into the microemulsion at the point of application, either during application (e.g. by co-application of a lipid/surfactant mixture and a solvent) or by adsorption of liquid (especially aqueous liquid) from the mucosal surface upon contact therewith. In this aspect, the "microemulsion" may be seen, in fact, as a "microemulsion precursor" but it will be evident that in both cases the result and functioning of the invention will be equivalent and thus both aspects are encompassed in the present description, where context allows. It will be necessary, however that where a "microemulsion precursor" is used, this will generate a true microemulsion as indicated herein, to at least a sufficient extent to provide the advantageous effects indicated.
The polar solvent or solvents, where present in the inventive microemulsion should comprise 5 to 95% (wt/wt), preferably 10 to 55% (wt/wt) of the same. It is also preferred that the at least one polar solvent has a pH exceeding pH 5.5.
The polar lipid in the microemulsion can be a suitable amphiphilic alcohol or amine, a sterol (e.g. cholesterol), a fat soluble vitamin, a glyceride, a phospholipid, a glycolipid, a sphingolipid, or any mixture thereof. Preferably, where present the glyceride is a mono-acyl glyceride, for example glyceryl monooleate, glyceryl monolinoleate or glyceryl monolinenoleate.
The polar lipid may comprise 1 to 99% (wt/wt), preferably 10 to 75% (wt/wt), most preferred 20 to 50% (wt/wt) of the microemulsion of the invention.
It is an important aspect of the invention that the microemulsion in itself, consisting of a non-polar lipid, at least one polar solvent, a surfactant, and a polar lipid as sole ingredients (optionally with inert carrier or excipients), provides an environment that can both wet and substantially enclose airborne particles.
The microemulsion of the invention should also comprise one or more non-polar lipids, such as di-acyl glycerides, triacyl glycerides, mineral oils, paraffin oils and esters, as well as ethers or waxes of fatty acids having a total number of carbon atoms more than 22. Preferably, the nonpolar lipid is a tri-acyl glyceride.
Excellent microemulsions are formed well by using non-polar oils, such as the normal alkanes, as the nonpolar lipid. Triglycerides are preferred because they are edible but other lipids may be used individually or as mixtures in any amount which can be adequately tollerated.
Evidently, components such as triglycerides may be made or modified synthetically or extensively purified. Alternatively and preferably, these may be used as available mixtures, such as those from natural sources. Natural sources include animal oils and plant/vegetable oils.
Acceptable animal oils include cod liver oil, lanolin oil, mink oil, orange roughy oil, and shark liver oil. Acceptable vegetable oils include almond, apricot kernel, avocado, castor, coconut, corn, evening primrose, jojoba, olive, safflower, sesame, soybean, and wheat germ oils. Some silicone oils are acceptable, such as certain linear polysiloxanes and cyclic dimethyl polysiloxanes such as cyclomethicone. It is preferred that the vegetable oil is sesame oil.
The non-polar lipid should comprise 1 to 99% (wt/wt), preferably 5 to 75% (wt/wt), most preferably 5 to 35% (wt/wt) of the microemulsion.
The microemulsion according to the invention can have a large number of applications where airborne particles have to be entrapped in the homogenous (microemulsion) film formed. These include different kinds of filter devices, which can be used for e.g. air cleaning and/or decontamination. However, the inventive emulsion is especially adapted for direct human use, i.e. to be applied to peripheral membrane linings of the nose, the eyes, the ears, the pharynx, and the larynx for entrapping airborne particles.
The microemulsions can be applied locally. Local applications include for example spray and drops, pipettes made up in single doses, a powder obtained by lyophilization of the emulsion, a tamponage, or a painting. A nasal pool device can be used no apply controlled amounts of the emulsion on a human nasal airway mucosa. Preferably, a mouth or nasal spray device is used, which contains the inventive microemulsion.
Where a powder is used, this may be conveniently generated by drying the microemulsions of the invention, which will thus form solvent-free or low-solvent microemulsion precursors. These precursors will again form (to at least some extent) the true microemulsions upon contact with, and incorporation of, aqueous fluids at the mucosal surface. Obviously, a convenient method for generating a dry powder microemulsion/precursor will be freeze-drying but the skilled worker will be aware of many equivalent techniques such as spray-drying which may be used to generate dry powders. Powders may also be milled and/or agglomerated to control particle sizes.
Where a powder is generated, it may also be advantageous to include other agents to improve the drying, the handling properties of the powder and/or the resuspension of the lipid components into a regenerated microemulsion. Suitable agents are generally water soluble and include polymers, salts and sugars.
The present invention focuses on, but is not limited to, those initial stages of the inflammatory process which take place at or in the peripheral membrane linings of the nose, the eyes, the ears, the pharynx, and the larynx. For example, at or in the nasal mucosa, an inflammation may result in increased levels of pro-inflammatory chemokines and cytokines, recruitment and activation of specific inflammatory cells, and changes in end-organ functions of the mucosa (e.g., the microcirculation, the secretory apparatus and the ciliary system). Inflammatory processes, although in part characterized by different inflammatory cells, are also associated with upper respiratory tract infections. In one aspect of the presention, there is thus provided a composition comprising a microemulsion of the invention plus an active agent. In particular, this active agent will generally be a pharmaceutical agent having a local effect upon these inflammatory cells, systems or symptoms. In a preferred embodiment the present invention thus provides a composition comprising a microemulsion of the invention and a locally acting drug.
Suitable locally acting drugs include anti-allergical drugs, such as glucocorticosteroids, antihistamines, leukotriene antagonists, chromoglycates, decongestants, such as alpha-agonists, anti-cholinergic drugs, and anti-infection drugs, such as antibiotics or anti-viral compounds. Particularly suitable drugs include budesonide, beclomethasone dipropionate and loratadine. Mixtures of such drugs are also suitable. Compositions of the present invention may require no additional carriers but, where desirable, any suitable pharmaceutically tollerable carrier or excipient may be included.

This patent is subject to a terminal disclaimer.

Microemulsions and its use for preventing airway diseases


Multivalent, multispecific molecules having at least one specificity for a pathogen and at least one specificity for the HLA class II invariant chain (Ii) are administered to induce clearance of the pathogen. In addition to pathogens, clearance of therapeutic or diagnostic agents, autoantibodies, anti-graft antibodies, and other undesirable compounds may be induced using the multivalent, multispecific molecules.

Most forms of nonsurgical cancer therapy, such as external irradiation and chemotherapy, are limited in their efficacy because of toxic side effects to normal tissues and cells, because of the limited specificity of these treatment modalities for cancer cells. This limitation is also of importance when anti-cancer antibodies are used for targeting toxic agents, such as isotopes, drugs, and toxins, to cancer sites, because, as systemic agents, they also circulate to sensitive cellular compartments such as the bone marrow. In acute radiation injury, destruction of lymphoid and hematopoietic compartments is a major factor in the development of septicemia and subsequent death.
In the field of organ transplantation, the recipient""s cellular immune response to the foreign graft is depressed with cytotoxic agents which affect the lymphoid and other parts of the hematopoietic system. Graft acceptance is limited by the tolerance of the recipient to these cytotoxic chemicals, many of which are similar to the anticancer (antiproliferative) agents. Likewise, when using cytotoxic antimicrobial agents, particularly antiviral drugs, or when using cytotoxic drugs for autoimmune disease therapy, e.g., in treatment of systemic lupus erythematosis, a serious limitation is the toxic effects to the bone marrow and the hematopoietic cells of the body.
The detection of a target site benefits from a high signal-to-background ratio of detection agent. Therapy benefits from as high an absolute accretion of therapeutic agent at the target site as possible, as well as a reasonably long duration of uptake and binding. In order to improve the targeting ratio and amount of agent delivered to a target site, the use of targeting vectors comprising diagnostic or therapeutic agents conjugated to a targeting moiety for preferential localization has long been known.
Examples of targeting vectors include diagnostic or therapeutic agent conjugates of targeting moieties such as antibody or antibody fragments, cell-or tissue-specific peptides, and hormones and other receptor-binding molecules. For example, antibodies against different determinants associated with pathological and normal cells, as well as associated with pathogenic microorganisms, have been used for the detection and treatment of a wide variety of pathological conditions or lesions. In these methods, the targeting antibody is directly conjugated to an appropriate detecting or therapeutic agent as described, for example, in Hansen et al., U.S. Pat. No. 3,927,193 and Goldenberg, U.S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,460,459, 4,460,561, 4,624,846 and 4,818,709, the disclosures of all of which are incorporated herein by reference.
One problem encountered in direct targeting methods, i.e., in methods wherein the diagnostic or therapeutic agent (the xe2x80x9cactive agentxe2x80x9d) is conjugated directly to the targeting moiety, is that a relatively small fraction of the conjugate actually binds to the target site, while the majority of conjugate remains in circulation and compromises in one way or another the function of the targeted conjugate. In the case of a diagnostic conjugate, for example, a radioimmunoscintigraphic or magnetic resonance imaging conjugate, non-targeted conjugate which remains in circulation can increase background and decrease resolution. In the case of a therapeutic conjugate having a very toxic therapeutic agent, e.g., a radioisotope, drug or toxin, attached to a long-circulating targeting moiety such as an antibody, circulating conjugate can result in unacceptable toxicity to the host, such as marrow toxicity or systemic side effects.
While some agents exist for clearing non-interacting agents, these agents do not generally involve the use of a specific mechanism for targeting clearance. In some cases, however, to speed up this hepatobiliary recognition process, the clearing agent may be substituted with sugar residues, primarily galactose, such that the galactosylated complex is recognized by the asialoglycoprotein receptors in the liver. By using a galactosylated biotin-protein, substantially all circulating streptavid-antibody and galactosylated biotin-protein is deposited into the liver on the first pass through, making the clearing process very fast and efficient. With circulating avidin conjugate removed, excess biotin-chelate-radionuclide is rapidly eliminated, preferably renally. Because the radionuclide spends a very short time in circulation, considerably less marrow toxicity to the patient is seen compared to when the radionuclide is attached directly to the antibody. However, it has been found that some of these clearing agents may bind to and remove the therapeutic or diagnostic agent that is beneficially bound to the target site.
U.S. Pat. No. 4,782,840 discloses a method for reducing the effect of elevated background radiation levels during surgery. The method is to inject the patient with antibodies specific for neoplastic tissue and which are labeled with radioisotopes having a suitably long half-life, such as Iodine-125. After injection of the radiolabeled antibody, the surgery is delayed at least 7-10 days, preferably 14-21 days, to allow any unbound radiolabeled antibody to be cleared to a low bloodpool, background level.
U.S. Pat. No. 4,932,412 discloses methods for reducing or correcting for non-specific background radiation during intraoperative detection. The methods include the administration to a patient who has received a radiolabeled primary antibody, of a contrast agent, subtraction agent or second antibody which binds the primary antibody.
A need exists, therefore, for improved clearing agents which work efficiently and rapidly to target the agent sought to be cleared to the cells responsible for clearance.
It is one object of the invention to provide multivalent molecules which are suitable for pharmaceutical applications that induce clearance of a variety of noxious substances. According to this and other objects of the invention, a multivalent molecule is provided which has at least one specificity for a noxious substance, such as a pathogenic organism, and at least one specificity for the HLA class II invariant chain (Ii). In one embodiment of the invention, the multivalent molecule is a multispecific antibody molecule. In another embodiment, at least one specificity is directed to a microorganism, such as a fungus. In still other embodiments, the pathogenic organism may be a cancer cell, a parasite or a virus, such as HIV. Further embodiments include pharmaceutical compositions of these multivalent molecules.
Another object of the invention is to provide multivalent molecules that are useful in treating sepsis. According to this an other objects of the invention, a multivalent molecule is provided which has at least one specificity for lipopolysaccharide (LPS) or lymphotoxin and at least one specificity for the HLA class II invariant chain (Ii). Pharmaceutical compositions are also provided.
Another object of the invention is to provide multivalent molecules that are useful in inducing clearance of excess diagnostic or therapeutic agents. According to this and other objects of the invention, a multivalent molecule is provided which has at least one specificity for a diagnostic or therapeutic agent and at least one specificity for the HLA class II invariant chain (Ii). Pharmaceutical compositions are also provided.
Another object of the invention is to provide multivalent molecules that are useful in inducing clearance of autoantibodies. According to this and other objects of the invention, a multivalent molecule is provided which has at least one specificity for a specific binding site of an autoantibody and at least one specificity for the HLA class II invariant chain (Ii).
Another object of the invention is to provide methods of treating a patient exposed to a pathogenic organism. The invention thus provides methods involving administering to the patient an effective amount of a multivalent molecule having at least one specificity for the pathogenic organism and at least one specificity for the HLA class II invariant chain (Ii). In different embodiments, the pathogenic organism is a cancer cell, a parasite or an infectious agent.
A further object of the invention is to provide methods of treating septic shock. According to this object, a method is provided for treating or preventing septic shock, which comprises administering to a patient an effective amount of a multivalent molecule having at least one specificity for lipopolysaccharide (LPS) or lymphotoxin and at least one specificity for the HLA class II invariant chain (Ii).
Still another object of the invention is to provide a method of inducing clearance of an excess diagnostic or therapeutic agent. According to this object, there is provided a method of inducing clearance of a therapeutic or diagnostic agent in a patient, in involves administering to the patient a multivalent molecule having at least one specificity for the agent and at least one specificity for the HLA class II invariant chain (Ii).
The invention relates, in general terms, to inducing clearance of a variety of noxious substances from the body. In one aspect of the invention, there is provided a multivalent therapeutic agent, which has at least two different binding specificities. A representative therapeutic agent contains at least one binding specificity for a noxious substance sought to be cleared and at least one specificity to the HLA class II invariant chain (Ii). In another aspect of the invention, there are provided methods of using these therapeutic agents to induce clearance in a patient.
Due to the multiple specificities of the subject therapeutic agents, it is likely that the inventive methods work by forming a bridge between the agent(s) sought to be cleared from the patient and HLA class II molecules. The resulting proximal association, in some manner, is believed to induce or facilitate internalization of the target agent, transportation into lysosomes and degradation of the agent therein.
As used herein xe2x80x9cclearancexe2x80x9d refers not only to the process of removing the target substance from the body, but also to earlier stages of this process. Clearance also refers to the sequestration of the target substance followed by the removal of the target from, for example, the circulation, lymphatic system, interstitial spaces and the body cavities.
Therapeutic Agents
The therapeutic agents of the invention are multivalent and multispecific. By xe2x80x9cmultivalentxe2x80x9d it is meant that the subject agents may bind more than one target, which may have the same or a different structure, simultaneously. A xe2x80x9ctargetxe2x80x9d is either the HLA class II invariant chain or an agent sought to be cleared. By xe2x80x9cmultispecificxe2x80x9d it is meant that the subject agents may bind simultaneously to at least two targets which are of different structure. For example, an agent having one specificity for HLA class II invariant chain and one other specificity for a pathogenic bacterium would be considered multivalent and multispecific because it can bind two structurally different targets simultaneously. On the other hand, a molecule having two specificities for HLA class II invariant chain, but no other specificities, would be multivalent but not multispecific.
Some preferred agents are bispecific, but in some cases additional specificities, e.g. two to six, are preferred. Similarly, some preferred agents are bivalent, but increasing the valency of the agent would be beneficial in binding either additional molecules of the same target or multiple different targets. On preferred class of agents, therefore is bispecific and is at least trivalent, having at least one binding site for Ii and two for the target molecule.
As indicated above, preferred therapeutic agents have at least one specificity directed to HLA class II invariant chain. This specificity confers on the therapeutic agent the characteristic of targeting to invariant chain-positive cells in many organs, such as liver, marrow, spleen, lymph nodes and skin. This targeting is associated with rapid clearance of the therapeutic agent containing this invariant chain specificity through internalization, transport to lysosomes, and subsequent degradation.
The at least one other specificity of the present therapeutic agents may be directed to nearly any substance which it is desirable to have cleared from the body. These substances may be, for example, toxins, pathogenic organisms (e.g., bacteria, fungi, and parasites), viruses, autoantibodies and chemotherapeutic agents. In one embodiment, the pathogenic organism is a fungus of the genus Cryptococcus, and especially Cryptococcus neoformans.
In another embodiment, the pathogenic organism to be cleared is a cancer cell. The cancer cell will be bound to a phagocytic cell expressing HLA class II invariant chain, which may be a macrophage, Kuppfer cell, or histiocyte, for example, with the subsequent destruction of the cancer cell by the phagocytic cell. Although the cancer cell may be internalized by the phagocytic cell, the cancer cell may first be killed by necrotic or apoptoic induction. The tumor-targeting moiety of the multispecific agent may be an antibody reactive with a tumor associated or tumor specific antigen.
Another substance which is desirable to have cleared from the body is an xe2x80x9cautoantibody,xe2x80x9d an antibody that recognizes a native epitope. Autoantibodies may form immune complexes with normal cells or serum components, leading to their damage or clearance by the immune system, just as immune complexes with foreign pathogens. One source of tissue damage is activation of the complement system. Most antibodies, including autoantibodies, have a site on the Fc portion of the immunoglobulin chain that can react with activated C1q or C3 components of the complement system. Complex formation between the autoantibody, activated C1q or C3, and the cellular surface initiates a cascading activation of other complement components, leading to the damage or destruction of the cell to which the autoantibody is bound.
As a result, autoantibodies are responsible for a large number of serious, sometimes life threatening, diseases. Beeson (1994) Am. J. Med. 96:457. For example, autoantibodies may recognize the acetylcholine receptors found in neural muscular junctions, for example. The resulting damage to muscular tissue leads to the development of myasthenia gravis. If the autoantibody is directed to platelets, the resulting platelet destruction can lead to chronic autoimmune thrombocytopenia purpura.
In one embodiment of the present invention, the at least one other specificity of the present therapeutic agent may be directed to the specific binding site of an autoantibody. By directing the other specificity to the specific binding site of the autoantibody, clearance of antibodies with potentially harmful specificities is achieved, while the clearance of useful, normal antibodies is avoided.
In another embodiment, the present therapeutic agent may induce clearance of autoantibodies in the form of an immune complex. In this case, the at least one other specificity of the present therapeutic agent preferably is directed to activated C1q, or activated C3 component, which is bound to the immune complex. Thus, autoantibody clearance may be induced regardless of the epitope recognized by the autoantibody specific binding site. By directing the other specificity only to complement components involved in immune complexes, the clearance and depletion of normal complement components may be avoided. Antibodies that specifically recognize immune complex-bound, activated C1q are described in U.S. Pat. No. 4,595,654, incorporated herein by reference.
Undesirable antibodies may also have specificities against transplanted tissue from other species. For example, porcine organs transplanted into humans typically complex with antibodies present within the human host within hours of transplantation. The host antibodies may activate the complement system, resulting in damage and rejection of the transplanted tissue, Fukushima et al. (1994) Transplantation 57:923; Pruitt et al., (1994) Transplantation 57:363. The major epitope on non-human tissue responsible for transplant rejection is the (xcex1-galactosyl epitope (Galxcex11-3Galxcex21-4GlcNAc-R). Galili et al. (1985) J. Exp. Med. 162:573. Up to 1% of all serum IgG in humans recognizes the xcex1-galactosyl epitope. Galili et al. (1984) J. Exp. Med. 160:1519.
It is one objective of the invention to ameliorate the rejection response of humans toward non-human tissue by inducing the clearance of antibodies directed toward transplanted tissue. In accord with this objective, the present therapeutic agent has at least one specificity directed to the specific binding site of an anti-graft antibody, and at least one specificity for Ii. In a preferred embodiment, the specific binding site of an anti-graft antibody recognizes the xcex1-galactosyl epitope. In a more preferred embodiment, the least one specificity for a specific binding site of the anti-graft antibody is a polymer of alpha-galactose.
The chemical constitution of the therapeutic agents may also vary, but they should be capable of specific binding. Accordingly, macromolecule, such as proteins, carbohydrates (e.g., lectins) and RNAs are preferred. Due to the well known ability to generate molecules capable of binding with a wide range of specificities, antibodies and antibody fragments and derivatives are particularly preferred. Both monoclonal and polyclonal antibodies may be prepared according to established methods in the art. Because they bind with a single, defined specificity, monoclonal antibodies are a preferred starting material. Having generated different monoclonal antibodies, and thus a variety different specificities, these starting molecules can be used to generate the multivalent, multispecific agents of the invention. The art is well versed in both recombinant and chemical methods (crosslinking) for generating such agents.
Fragments of antibodies include any portion of the antibody which is capable of binding the target antigen. Antibody fragments specifically include F(abxe2x80x2)2, Fab, Fabxe2x80x2 and Fv fragments. These can be generated from any class of antibody, but typically are made from IgG or IgM. They may be made by conventional recombinant DNA techniques or, using the classical method, by proteolytic digestion with papain or pepsin. See CURRENT PROTOCOLS IN IMMUNOLOGY, chapter 2, Coligan et al., eds., (John Wiley and Sons 1991-92).
F(abxe2x80x2)2 fragments are typically about 110 kDa (IgG) or about 150 kDa (IgM) and contain two antigen-binding regions, joined at the hinge by disulfide bond(s). Virtually all, if not all, of the Fc is absent in these fragments. Fabxe2x80x2 fragments are typically about 55 kDa (IgG) or about 75 kDa (IgM) and can be formed, for example, by reducing the disulfide bond(s) of an F(abxe2x80x2)2 fragment. The resulting free sulfhydryl group(s) may be used to conveniently conjugate Fabxe2x80x2 fragments to other molecules, such as detection reagents (e.g., enzymes).
Fab fragments are monovalent and usually are about 50 kDa (from any source). Fab fragments include the light (L) and heavy (H) chain, variable (VL and VH, respectively) and constant (CL and CH, respectively) regions of the antigen-binding portion of the antibody. The H and L portions are linked by an intramolecular disulfide bridge.
Fv fragments are typically about 25 kDa (regardless of source) and contain the variable regions of both the light and heavy chains (VL and VH, respectively). Usually, the VL and VH chains are held together only by non-covalent interacts and, thus, they readily dissociate. They do, however, have the advantage of small size and they retain the same binding properties of the larger Fab fragments. Accordingly, methods have been developed to crosslink the VL and VH chains, using, for example, glutaraldehyde (or other chemical crosslinkers), intermolecular disulfide bonds (by incorporation of cysteines) and peptide linkers. The resulting Fv is now a single chain (i.e., SCFv).
One preferred method involves the generation of SCFvs by recombinant methods, which allows the generation of Fvs with new specificities by mixing and matching variable chains from different antibody sources. In a typical method, a recombinant vector would be provided which comprises the appropriate regulatory elements driving expression of a cassette region. The cassette region would contain a DNA encoding a peptide linker, with convenient sites at both the 5xe2x80x2 and 3xe2x80x2 ends of the linker for generating fusion proteins. The DNA encoding a variable region(s) of interest may be cloned in the vector to form fusion proteins with the linker, thus generating an SCFv.
In an exemplary alternative approach, DNAs encoding two Fvs may be ligated to the DNA encoding the linker, and the resulting tripartite fusion may be ligated directly into a conventional expression vector. The SCFv DNAs generated any of these methods may be expressed in prokaryotic or eukaryotic cells, depending on the vector chosen.
In one embodiment, the agent sought to be cleared is a parasite, such as an leishmania, malaria, trypanosomiasis, babesiosis, or schistosomiasis. In such cases the inventive molecules may be directed against a suitable parasite-associated epitope which includes, but is not limited to, the following.
In another embodiment, the agent sought to be cleared is a virus, such as human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), or hepatitis. In such cases, the inventive therapeutic agent may be directed against a suitable viral epitope including, but not limited to:
The agent sought to be cleared may also be bacterial. In this case, the inventive molecule may have a specificity to a suitable bacterial epitope which includes, but is not limited to:
Pharmaceutical Compositions
Pharmaceutical compositions according to the invention comprise at least one therapeutic agent as described above. In addition, these compositions typically further contain a suitable pharmaceutical excipient. Many such excipients are known to the art and examples may be found in REMINGTON""S PHARMACEUTICAL SCIENCES, chapters 83-92, pages 1519-1714 (Mack Publishing Company 1990) (Remington""s), which are hereby incorporated by reference. The choice of excipient will, in general, be determined by compatibility with the therapeutic agent(s) and the route of administration chosen. Although the subject compositions are suitable for administration via numerous routes, parenteral administration is generally preferred. The inventive compositions may be formulated as a unit dose which will contain either a therapeutically effective dose or some fraction thereof.
Methods of Preparing Multivalent Molecules
Multivalent, multispecific antibody derivatives can be prepared by a variety of conventional procedures, ranging from glutaraldehyde linkage to more specific linkages between functional groups. The antibodies and/or antibody fragments are preferably covalently bound to one another, directly or through a linker moiety, through one or more functional groups on the antibody or fragment, e.g., amine, carboxyl, phenyl, thiol, or hydroxyl groups. Various conventional linkers in addition to glutaraldehyde can be used, e.g., disiocyanates, diiosothiocyanates, bis(hydroxysuccinimide) esters, carbodiimides, maleirmidehydroxy-succinimde esters, and the like. The optimal length of the linker may vary according to the type of target cell. The most efficacious linker size can be determined empirically by testing (and ensuring) reactivity to both target and Ii. Such immunochemical techniques are well known.
A simple method to produce multivalent antibodies is to mix the antibodies or fragments in the presence of glutaraldehyde. The initial Schiff base linkages can be stabilized, e.g., by borohydride reduction to secondary amines. A diiosothiocyanate or carbodiimide can be used in place of glutaraldehyde as a non-site-specific linker.
The simplest form of a multivalent, multispecific antibody is a bispecific antibody comprising binding specificities both to a target agent to be cleared and to Ii. Bispecific antibodies can be made by a variety of conventional methods, e.g., disulfide cleavage and reformation of mixtures of whole IgG or, preferably F(abxe2x80x2)2 fragments, fusions of more than one hybridoma to form polyomas that produce antibodies having more than one specificity, and by genetic engineering. Bispecific antibodies have been prepared by oxidative cleavage of Fabxe2x80x2 fragments resulting from reductive cleavage of different antibodies. This is advantageously carried out by mixing two different F(abxe2x80x2)2 fragments produced by pepsin digestion of two different antibodies, reductive cleavage to form a mixture of Fabxe2x80x2 fragments, followed by oxidative reformation of the disulfide linkages to produce a mixture of F(abxe2x80x2)2 fragments including bispecific antibodies containing a Fabxe2x80x2 portion specific to each of the original epitopes (i.e., target and li). General techniques for the preparation of multivalent antibodies may be found, for example, in Nisonhoff et al., Arch Biochem. Biophys. 93: 470 (1961), Hammerling et al., J. Exp. Med. 128: 1461 (1968), and U.S. Pat. No. 4,331,647.
More selective linkage can be achieved by using a heterobifunctional linker such as maleimide-hydroxysuccinimide ester. Reaction of the ester with an antibody or fragment will derivatize amine groups on the antibody or fragment, and the derivative can then be reacted with, e.g., an antibody Fab fragment having free sulfhydryl groups (or, a larger fragment or intact antibody with sulfhydryl groups appended thereto by, e.g., Traut""s Reagent). Such a linker is less likely to crosslink groups in the same antibody and improves the selectivity of the linkage.
It is advantageous to link the antibodies or fragments at sites remote from the antigen binding sites. This can be accomplished by, e.g., linkage to cleaved interchain sulfydryl groups, as noted above. Another method involves reacting an antibody having an oxidized carbohydrate portion with another antibody which has at lease one free amine function. This results in an initial Schiff base (imine) linkage, which is preferably stabilized by reduction to a secondary amine, e.g., by borohydride reduction, to form the final product. Such site-specific linkages are disclosed, for small molecules, in U.S. Pat. No. 4,671,958, and for larger addends in U.S. Pat. No. 4,699,784.
The interchain disulfide bridges of the an F(abxe2x80x2)2 fragment having target specificity are gently reduced with cysteine, taking care to avoid light-heavy chain linkage, to form Fabxe2x80x2-SH fragments. The SH group(s) is(are) activated with an excess of bis-maleimide linker (1,1xe2x80x2-(methylenedi-4,1-phenylene)bis-malemide). An Ii-specific Mab, such as LL1, is converted to Fabxe2x80x2-SH and then reacted with the activated target-specific Fabxe2x80x2-SH fragment to obtain a bispecific antibody.
Alternatively, such bispecific antibodies can be produced by fusing two hybridoma cell lines that produce anti-target Mab and anti-li Mab. Techniques for producing tetradomas are described, for example, by Milstein et al., Nature 305: 537 (1983) and Pohl et al., Int. J. Cancer 54: 418 (1993).
Finally, such bispecific antibodies can be produced by genetic engineering. For example, plasmids containing DNA coding for variable domains of an anti-target Mab can be introduced into hybridomas that secrete LL1 antibodies. The resulting xe2x80x9ctransfectomasxe2x80x9d produce bispecific antibodies that bind target and Ii. Alternatively, chimeric genes can be designed that encode both anti-target and anti-Ii binding domains. General techniques for producing bispecific antibodies by genetic engineering are described, for example, by Songsivilai et al., Biochem. Biophys. Res. Commun. 164: 271 (1989); Traunecker et al., EMBO J. 10: 3655 (1991); and Weiner et al., J. Immunol. 147: 4035 (1991).
A higher order multivalent, multispecific molecule can be obtained by adding various antibody components to a bispecific antibody, produced as above. For example, a bispecific antibody can be reacted with 2-iminothiolane to introduce one or more sulfhydryl groups for use in coupling the bispecific antibody to an further antibody derivative that binds an the same or a different epitope of the target antigen, using the bis-maleimide activation procedure described above. These techniques for producing multivalent antibodies are well known to those of skill in the art. See, for example, U.S. Pat. No. 4,925,648, and Goldenberg, international publication No. WO 92/19273, which are incorporated by reference.
Methods of Treatment
The methods of the invention typically involve administering to a patient in need of treatment a therapeutically effective amount of a composition which comprises a therapeutic agent of the invention. The patient is usually human, but may be a non-human animal. A patient will be in need of treatment where it is desirable to induce clearance of a target agent.
A therapeutically effective amount is generally an amount sufficient to accelerate clearance of the target agent versus a control.
Some methods involve the use of the instant therapeutic agents to induce clearance of cytoreductive agents (chemotherapeutic agents). In a typical method, a patient is treated with a cytoreductive agent, then the excess cytoreductive agent is removed by administration of an inventive compound having at least one specificity for the cytoreductive agent. In one exemplary method, the cytoreductive agent comprises an antibody for targeting and the inventive compound has a specificity for the antibody portion of the agent. In this manner, any portion of the cytoreductive agent that fails to specifically interact with its target is removed. It is anticipated that this method will allow the use of higher, more effective doses of the cytoreductive agent. Side effects will be minimized because the inventive compounds induce clearance of any excess.
Other methods involve reducing the background caused by excess non-localizing diagnostic agents. These methods are useful, for example, in an imaging procedure where a targeting agent (e.g., an antibody) is conjugated with a detectable marker (e.g., a radionuclide). In a typical method, the diagnostic agent would be administered to a patient and, following administration but before detection, an inventive compound having at least one specificity for the diagnostic agent and at least one specificity for Ii is provided to the patient, thereby inducing clearance of the excess diagnostic.
Still other methods involved the clearance of pathogens, such as bacteria, from the patient. It is envisioned that this approach will have particular benefit for patients who have become septic. Typically, these methods involve administering an inventive compound having at least one specificity for a pathogen of interest (e.g., endotoxin) and at least one specificity for Ii, thereby inducing clearance of the pathogen.
The term xe2x80x9ctreatingxe2x80x9d in its various grammatical forms in relation to the present invention refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a disease state, disease progression, disease causative agent (e.g., bacteria or viruses) or other abnormal condition. Because some of the inventive methods involve the physical removal of the etiological agent, the artisan will recognize that they are equally effective in situations where the inventive compound is administered prior to, or simultaneous with, exposure to the etiological agent (prophylactic treatment) and situations where the inventive compounds are administered after (even well after) exposure to the etiological agent.