Methods and apparatus are described for selecting operation devices or hardware components for a processor, such as an embedded processor having pipelined data paths. The process may include identifying a set of hardware components, such as function units, and a plurality of characteristics for those hardware components. A first set of characteristics may include the ability to add, subtract, multiply, and the like, or they may be multi-functional. A second set of characteristics for the hardware components may include cost, throughput and the like. A plurality of these characteristics of the hardware components are incorporated into an algorithm, which is then solved for one or more desired parameters, such as type and number of hardware components. In one preferred embodiment, the least cost assembly of hardware components is found for carrying out a set of computations defined by an algorithm to be executed on the processor according to a preferred initiation interval.

These inventions relate to processor design, and more specifically to allocation of function units in processor design, such as for systolic processors and application specific integrated processors (ASIPs).
Processor design is a very time intensive and expensive process. For new and unique processor designs, no automated design techniques exist for selecting and designing the mix of processor components that would be incorporated into the final processor design. While there exist algorithms incorporated into software packages that can help in designing new processors, such software packages do not give a result which is a final design, let alone an optimal design. Typically, those software packages provide approximate solutions to a design problem, typically leading to additional design effort and over-design to account for the lack of precision in those software packages. Additionally, the design process may start entirely from scratch, which would result in substantial time being consumed analyzing possible design configurations before designing the details of the processor. On the other hand, designing a new processor using preexisting designs necessarily incorporates the design benefits and flaws of the preexisting design, which may or may not be acceptable or optimal for the new design.
All conventional processor design software packages are heuristic in nature. In other words, they rely on design criteria and/or methods that in the past have proven more effective than other criteria or methods. However, in order to apply to more than one processor design or design methodology, such design criteria and methods must be sufficiently general to provide predictable results. Therefore, such heuristic software packages provide relatively high-level solutions without a complete contribution to details of the design. Additionally, heuristic software packages necessarily lead to significant trial and error in an attempt to optimize the processor design. Consequently, design of new processors is time intensive and expensive.
Processors are often designed to incorporate pipelined data paths to speed processing throughput, reduce initiation intervals and to optimize use of the various function units, such as adders, multipliers, comparators, dividers and the like. These data paths are formed from an interconnected assembly of function units and register files. The function units and register files may be interconnected by busses. Because these data paths may include a large number of function units, register files and bus segments, the job of selecting the function units, register files and bus segments is very difficult, to say the least. The task is made more difficult if one desires to find the optimal configuration of these units having the lowest or a low cost relative to other configurations. Because of the huge number of possible configurations, it is difficult if not impossible to find optimal configurations.
Pipelined data paths are particularly useful in processing iterative instructions, such as those found in instruction loops, and especially nested instruction loops. When considering a subset of situations where the instruction loops are known, such as those used with embedded processors, the task of designing the optimal, low-cost processor still exists because of the large number of different function units, register configurations and bus configurations that are possible. Heuristic software design solutions used for designing processors are not suitable for finding solutions to such multi-dimensional problems. Because there are so many variables to consider, it is too difficult to optimize all of variables to arrive at a suitable solution without great expenditure of time and effort.
The present inventions provide methods and apparatus for more easily and efficiently producing computing systems, for example those incorporating processor arrays having processor elements with function or execution units, register files, busses, and the like. These methods and apparatus reduce the time required for designing these processors, and reduce the amount of trial and error used in processor design. They also find a better combination of hardware components, as tested by one or more quantifiable parameters, than those developed using heuristic methods, leading to overall superior results. The methods and apparatus may provide solutions to function unit allocation problems, or at least a starting point for allocating function units in processor design. They also eliminate the costs associated with starting the design of a new processor from scratch, which often may be necessary in the design of embedded processors, and they allow the more time intensive design process to start later in the conventional processor design flow.
These and other aspects of the present inventions are provided by methods and apparatus for selecting operation devices or hardware components for a processor, such as for an embedded processor having pipelined data paths. The hardware components could be function units, registers, busses, or other items that could be incorporated into a processor. The apparatus can take any number of forms, including computers and other processors, such as mainframes, workstations, and the like, as well as apparatus containing instructions or data for use in controlling such processors, such as disk drives, removable storage media, and temporary storage. In one aspect of the present inventions, the process includes identifying a set of hardware components, such as function units, and a plurality of characteristics for those hardware components. A first set of characteristics may include a repertoire, such as the ability to add, subtract, multiply, and the like, and a second set of characteristics for the hardware components may include the number of cycles used for a given operation for the particular hardware component (data interval, i.e. the number of time slots or cycles in each unit of type i for an operation of type j), cost and the like. A plurality of these characteristics of the hardware components are incorporated into an algorithm, which is then solved for one or more desired parameters, such as type and number of hardware components.
In one preferred embodiment, the algorithm is an integer linear program which is loaded with a list of the set of function units available for incorporation into an embedded processor, the cost of each of the function units, the data intervals associated with the function units, as well as any other necessary data associated with the function units. The integer linear program is also preferably given one or more constraints, such as the number of each of the function units must be an integer. Other constraints include the requirement that the sum over all of the function units of the number of cycles carried out in a function unit for its operations divided by the data intervals is at least the number of operations of a given type. The integer linear program may also be given bounds such as a maximum cost for the result, or other constraints based on input from a user or designer. Integer linear programs can quickly and efficiently provide a desired solution or an approximation to a solution. Where the result is a desired solution, the result can be used to design a complete processor and provide sufficient information to produce a hardware description expressed in a hardware design language such as VHDL. Where the result is an approximation to a solution, the designer can use that result to more quickly design a processor according to the defined design criteria for the processor.
Integer linear programs are particularly useful for providing solutions to multi-dimensional problems, and are particularly appropriate for allocating function units in processor elements of synchronous processor arrays. The possible combinations of function units are so numerous that optimal selection of function units for a particular processor design may be effectively impossible. This is especially the case with multi-function function units, where because of a particular combination of operations a multi-function function unit would serve better than a number of discrete function units carrying out the same operations. Algorithms used to solve an integer linear program are well known and produce reliable results.
In a further preferred form of the invention, the integer linear program is configured to minimize cost of the function units while still ensuring that all operations for a given set of instructions within a loop are included. Using an integer linear program to minimize cost is especially attractive for allocating function units in an embedded processor using data pipelining. Embedded processors will be carrying out predefined operations, many of which will be repetitive loop operations. Consequently, there is not as much flexibility necessary in the function units to accommodate a variety of different operations. Thus, the focus in solving the integer linear program may be on minimizing the cost of the allocation of function units knowing that a number of different function unit combinations are available for carrying out any given set of operations. While parameters other than cost can be optimized, minimizing cost for embedded processors is particularly attractive in view of the expected proliferation of embedded processors in equipment, appliances and other apparatus.
In another form of the inventions, an upper bound on the cost may be input to the integer linear program. Such additional input or constraint effectively limits the number of possible solutions to the integer linear program. If a given constraint would be violated by a particular set of combinations, the integer linear program can easily eliminate such combinations without otherwise testing whether or not the particular set of combinations meet all other criteria. Other constraints can also be imposed on the integer linear program based on heuristics known to the designer.

Function unit allocation in processor design


A dual component oral composition to suppress oral malodor and provide long lasting breath protection in the oral cavity which comprises a first dentifrice component having a substantially neutral pH and containing a zinc ion releasable compound and a chlorite ion releasable compound, the second dentifrice component having an acid pH, the first and second components being maintained separate from each other until dispensed and combined for application to teeth, the combined components having a pH of no greater than 7.

1. Field of the Invention
The present invention relates to a dentifrice composition useful for reducing oral malodor, and more particularly to a dual component dental composition containing zinc and chlorite ion releasable compounds.
2. The Prior Art
xe2x80x9cOral compositionxe2x80x9d means a composition for topical applications to the oral cavity to clean and care for the teeth as well as the oral cavity surfaces. Representative of such compositions are oral hygiene products for delivering therapeutic and cosmetic benefits to the oral cavity such as mouthwashes or rinses, toothpaste, dental gels, tooth powder, chewing gum, lozenges, strips and similar products. The benefits delivered by oral compositions include the suppression of dental calculus formation and the prevention of dental disorders such as caries, periodontitis and gingivitis as well as the elimination of halitosis.
Dental plaque is a deposit which forms on teeth and consists of inorganic and organic components derived from saliva, food and bacteria which are present in the oral cavity. When plaque undergoes calcification it forms dental calculus composed largely of calcium and orthophosphate arranged in a crystal lattice called hydroxyapatite.
Oral malodor, clinically referred to as halitosis, is caused by the putrefactive activity of microorganisms on appropriate substrate components of dental plaque, debris adhering to mucous membranes and salivary cellular elements to produce volatile sulfur compounds primarily hydrogen sulfide, methyl mercaptan and traces of methyl sulfide.
Methods of inhibiting volatile sulfur compounds to reduce the production of mouth odor have included the use of oral compositions such as toothpastes and mouthrinses containing chlorite ions or zinc ions have been disclosed by the prior art, as for example, U.S. Pat. No. 6,132,702. U.S. Pat. No. 4,992,259. U.S. Pat. No. 5,753,217 discloses a method of reducing oral malodor comprising preparing a solution of sodium chlorite and a zinc salt and applying the solution as a mouth rinse, wherein the sodium chlorite concentration in the solution is between 500 and 1000 parts per million (ppm) and wherein the concentration of Zn ion in the solution is between 200 and 500.
Although useful as anti-malodor agents, applicants assays of the efficacy of chlorite ion releasable compounds as well as zinc ion releasable compounds have indicated that such compounds are not fully effective in suppressing oral malodor, and that efficacy improvements are necessary for full acceptance by consumers of oral care products containing these agents.
In accordance with the present invention there is provided a method to suppress oral malodor to provide long lasting breath protection wherein there is applied to the oral cavity a multicomponent dentifrice comprised of two separately housed, semi-solid aqueous components; the first component containing a zinc salt as the source of zinc ions and a chlorite salt as a source of chlorite ions in an orally acceptable vehicle having a substantially neutral pH of about 6.0 to 7.5 and the second component having an acid pH of from about 2.0 to about 6.0, whereby upon mixing and combination of the two components the pH of the final dentifrice is no greater than 6.5 with the generation of chlorine dioxide whereby there is effected an unexpected reduction in breath volatile sulfur compounds (VSC) responsible for breath malodor.
In use, the components of the two component dentifrice of the present invention comprise a first substantially neutral pH dentifrice component, and a second acidic dentifrice component. The two components are preferably formulated with water, humectants, surfactant and abrasive to have similar physical characteristics, so that the two components may be simultaneously delivered in desired predetermined amounts by extrusion when separately housed in a multicompartmented container such as a tube or pump device.
The neutral pH dentifrice component of the present invention, has a pH of about 6.0 to 7.5 and preferably about 6.8. The acidic dentifrice component is prepared using a similar vehicle to which an acid compound has been added to adjust the pH of such dentifrice component in the acid range of about 2.0 to about 6.0 and preferably about 4.0 to about 5.5. The pH of the final dentifrice after components are combined for use is no greater than 6.5 and preferably between about 5.8 and about 6.4.
The humectant used in the preparation of the dentifrice components is generally a mixture of humectants, such as glycerol, sorbitol and a polyethylene glycol of molecular weight in the range of 200 to 1000, but other mixtures of humectants and single humectants may also be employed. The humectant content is in the range about of 10% to about 80% by weight and preferably about 20 to about 50% by weight of the dentifrice component. The water content is in the range of about 10 to about 40% by weight and preferably about 20 to about 30% by weight.
Thickeners include organic and inorganic thickeners. Inorganic thickeners which may be included in the dentifrice components include amorphous silicas such as Zeodent 165 available from Huber Corporation, and Sylox 15 from W. R. Grace.
Organic thickeners of natural and synthetic gums and colloids may also be used to prepare the dentifrice components of the present invention. Examples of such thickeners are carrageenan (Irish moss), xanthan gum, sodium carboxymethyl cellulose, starch, polyvinylpyrrolidone, hydroxyethylpropylcellulose, hydroxybutyl methyl cellulose, hydroxypropyl methyl cellulose, and hydroxycthyl cellulose.
The inorganic thickener may be incorporated in the dentifrice components of the present invention at a concentration of about 0.5 to about 5% by weight and preferably about 1 to about 3% by weight. The organic thickener may be incorporated in the compositions of the present invention at a concentration of about 0.1 to about 3% by weight and preferably about 0.4 to about 1.5% by weight.
Surfactants may be incorporated in the dentifrices to provide foaming properties. The surfactant material is preferably anionic or nonionic in nature. Suitable examples of anionic surfactants arc higher alkyl sulfates such as potassium or sodium lauryl sulfate which is preferred, higher fatty acid monoglyceride monosulfates, such as the salt of the monosulfated monoglyceride of hydrogenated coconut oil fatty acids, alkyl aryl sulfonates such as sodium dodecyl benzene sulfonate, higher fatty sulfoacetates, higher fatty acid esters of 1,2 dihydroxy propane sulfonate.
The surfactant is generally present in the dentifrice component compositions of the present invention at a concentration of about 0.5 to about 10.0% by weight and preferably about 1.0 to about 5.0% by weight.
Abrasives may be incorporated in the dentifrice components of the present invention and preferred abrasives are siliceous materials, such as silica. A preferred silica is a precipitated amorphous hydrated silica, such as Sorbosil AC-35, marketed by Crosfield Chemicals, or Zeodent 115 from Huber Company but other abrasives may also be employed, including hydroxyapatite, sodium metaphosphate, potassium metaphosphate, tricalcium phosphate, calcium phosphate dihydrate, anhydrous dicalcium phosphate, calcium pyrophosphate, magnesium orthophosphate, trimagnesium phosphate, calcium carbonate, sodium bicarbonate, alumina trihydrate, aluminum silicate, calcined alumina and bentonite.
The concentration of abrasive in the dentifrice component compositions of the present invention will normally be in the range of 2 to about 40% by weight and preferably 5 to 25% by weight.
Zinc ion releasable compounds useful in the practice of the present invention are generally water soluble zinc salts including zinc nitrate, zinc citrate, zinc chloride, zinc sulfate, zinc bicarbonate and zinc oxalate with zinc nitrate being preferred. The zinc salt is generally incorporated in the neutral pH dentifrice component at a concentration of about 0.25 to about 10% by weight and preferably about 0.5 to about 2% by weight.
Chlorite ion releasable compounds include alkali metal chlorites, alkaline earth metal chlorites, and any other transition metals, inner transition metal chlorites and/or polymeric salts. Water soluble chlorite salts are preferred. Examples of suitable metal chlorites include calcium chlorite, barium chlorite, magnesium chlorite, lithium chlorite, sodium chlorite and potassium chlorite. Sodium chlorite and potassium chlorite are preferred. Sodium chlorite is particularly preferred. Mixtures of two or more sources of chlorite may also be used. The chlorite ion releasable salt is generally incorporated in the neutral pH dentifrice component at a concentration of about 0.5 to abut 5% by weight and preferably about 0.1 to about 1% by weight.
The acidic dentifrice component of the dentifrice composition of the present invention, which is maintained physically separate from the neutral dentifrice component until mixing before use, contains an acid or mixture of acids, to acidulate and thereby activate the chlorite compound present in the neutral dentifrice component to release chlorine dioxide, when the two components arc combined prior to use.
Acidic compounds which can be present in the acidic dentifrice component of the present invention include both mineral and organic acids, such as, sulfuric acid, hydrochloric acid, malic acid, alginic acid, citric acid, succinic acid, lactic acid, tartaric acid, potassium bitartrate, acid sodium citrate, phosphoric acid, and sodium acid phosphate. Acid phosphates are preferred, including phosphoric acid, or salts of phosphoric acid containing the PO4 ion, as such acids or acid salts thereof, such as sodium phosphate monobasic, not only provide the necessary acidity, but also provide phosphate ions, to inhibit any tooth enamel demineralization which may occur with the application of the two component dentifrice to the teeth. The preferred acid, phosphoric acid, is commercially available as a liquid at 85% concentration. The acid is added to the dentifrice component in an amount to maintain the pH of the dentifrice at a pH of about 2.0 to about 6.0 and preferably about 4.0 to about 5.5 when the neutral and acidic dentifrice components of the present invention are combined, the pH of the combined compositions is between about 5.8 to about 6.4.
Fluoride providing salts having anticaries efficacy may also be incorporated in the dentifrice of the present invention and are characterized by their ability to release fluoride ions in water. It is preferable to employ a water soluble fluoride salt providing about 10-2,000 ppm of fluoride ion, and preferably about 1000-1500 ppm of fluoride ion. Among these materials are water soluble inorganic metal salts, for example, sodium fluoride, potassium fluoride, sodium monofluorophosphate, stannous fluoride and sodium fluorosilicate. Sodium fluoride, sodium monoflurophosphate and stannous fluoride are preferred fluoride providing salts.
Pyrophosphate salts having anticalculus efficacy useful in the practice of the present invention include water soluble salts such as dialkali or tetraalkali metal pyrophosphate salts such as Na4P2O7(TSPP), K4P2O7, Na2K2P2O7, Na2H2P2O7 and K2H2P2O7. Polyphosphate salts may include the water soluble alkali metal tripolyphosphates such as sodium tripolyphosphate and potassium tripolyphosphate.
The pyrophosphate salts are incorporated in a dentifrice component of the present invention at a concentration of about 0.05 to about 2.0% by weight, and preferably about 0.5 to about 2% by weight. Polyphosphate salts can be incorporated in the dentifrice composition of the present invention at a concentration of about 1.0 to about 7.0% by weight.
Colorants such as pigments and dyes may be used in the practice of the present invention. Pigments include nontoxic, water insoluble inorganic pigments such as titanium dioxide and chromium oxide greens, ultramarine blues and pinks and ferric oxides as well as water insoluble dye lakes prepared by extending calcium or aluminum salts of FDandC dyes on alumina such as FDandC Green #1 lake, FDandC Blue #2 lake, FDandC RandD #30 lake and FDandC #Yellow 15 lake. The pigments have a particle size in the range of 5-1000 microns, preferably 250-500 microns, and are present at a concentration of 0.5 to 3% by weight.
Dyes used in the practice of the present invention are generally food color additives presently certified under the Food Drug and Cosmetic Act for use in the food and ingested drugs, including dyes such as FDandC Red No. 3 (sodium salt of tetraiodofluorescein), FDandC Yellow No. 5 (sodium salt of 4-p-sulfophenylazo-1-p-sulfophenyl-5-hydroxypyrazole-3 carboxylic acid), FDandC Yellow No. 6 (sodium salt of p-sulfophenylazo-B-naphtol-6-monosulfonate), FDandC Green No. 3 (disodium slat of 4-{[4-(N-ethyl-p-sulffobenzylamino)-phenyl]-(4-hydroxy-2-sulfoniumphenyl)-mewthylene}-[1-(N-ethyl-N-p-sulfobenzyl)-xcex94-3,5-cyclohexadienimine], FDandC Blue No. 1 (disodium salt of dibenzyldiethyldiaminotriphenylcarbinol trisulfonic acid of indigotin) and mixtures thereof in various proportions. The concentration of the dye for the most effective result in the present invention is present in the dentifrice composition in an amount from about 0.0005 percent to about 2 percent of the total weight.
A striped dentifrice product may be obtained using the dual component dentifrice of the present invention, wherein colorants of contrasting colors arc incorporated in each of the dentifrice components to be dispensed; the colorants being pharmacologically and physiologically non-toxic when used in the suggested amounts. Colorants used in the practice of the present invention include both the pigments and dyes discussed above.
Any suitable flavoring or sweetening material may also be incorporated in the dentifrice composition of the present invention. Examples of suitable flavoring constituents arc flavoring oils, e.g., oils of spearmint, peppermint, wintergreen, sassafras, clove, sage, eucalyptus, marjoram, cinnamon lemon, and orange, and methyl salicylate. Suitable sweetening agents include sucrose, lactose, maltose, sorbitol, xylitol, sodium cyclamate, perillatine, and sodium saccharin. Suitably, flavor and sweetening agents may together comprise from 0.01% to 5% or more of the preparations.
To prepare either of the dentifrice components of the present invention, generally the humectants e.g. glycerin, propylene glycol, polyethylene glycol are used to disperse with any organic thickeners, pigments such as titanium dioxide and any polyphosphates included as anti-calculus ingredients. Water is then added into this dispersion followed by sweetener. In the first component, the zinc and chlorite salts as well as a fluoride ion source is added. In the second component an ingredient to lower the pH to an acid level is added, such as phosphoric acid. These ingredients are mixed until a homogenous phase is obtained for each component. Thereafter inorganic thickener, silica abrasive, flavor and surfactant ingredients are added and the ingredients mixed at high speed under vacuum of from about 20 to 100 mm of Hg. The resultant product in the case of each component is a homogeneous, semi-solid, extrudible paste product.
The multicomponent dentifrice composition of the present invention is packaged in a suitable dispensing container in which the components are maintained physically separated and from which the separated components may be dispensed synchronously as a combined ribbon for application to a toothbrush. Such containers arc known in the art. An example of such a container is a two compartment dispensing container, such as a tube, having collapsible sidewalls, as disclosed in U.S. Pat. Nos. 4,487,757, 4,687,663 and 5,954,234; wherein, the tube body is formed from a collapsible plastic web such as polyethylene or polypropylene and is provided with a partition within the container body defining separate compartments in which the physically separated components are stored and from which they are dispensed through a suitable dispensing outlet.
The following Examples are further illustrative of the present invention, but it is understood that the invention is not limited thereto. All amounts and proportions referred to herein and in the appended claims are by weight, unless otherwise stated.

Dual component dentifrice for reducing mouth odors


This invention relates to chewable objects for animals which contain, as a dentally therapeutic ingredient, one or more cationic substances. The inventive therapeutic animal chews are of sufficient durability to allow for a chewing cycle long enough for the release of the aforementioned cationic substances into saliva. Furthermore, the inventive animal chews may contain an effective amount of a counter-ionic compound, such as an alkali metal salt, to allow for rapid solubilization of said cationic antimicrobial substance into the saliva of an animal chewing thereupon, especially when delivered or carried on a carrier having a negatively charged surface.

The invention is directed to methods and compositions for delivery of therapeutic agents to an animal by oral administration.
It is known that the dental health of dogs or other domestic animals is often deficient owing to the impossibility of achieving an effective brushing of their teeth with suitable dental products in a way comparable to that in humans.
A number of therapeutic proteinaceous animal chews have been described in the prior art to address this problem. The therapeutic effect from these prior art compositions or devices comes primarily from the physical act of chewing an object to provide an abrasive effect on the teeth prior to swallowing the compositions. The act of regularly chewing an object (such as rawhide) sufficiently rigid to allow for an oral residence time of greater than thirty seconds or so has been shown to result in reduced tartar accumulation compared to a quickly consumable object, such as a biscuit (Lags, et al J. Am. Veterinary Medical Ass, 197, pp 213-219 (1990)).
Simone et al. U.S. Pat. No. 5,296,209, reported on an attempt to clean an animal""s teeth through providing a foodstuff with a texture that allows for the animals tooth, during the act of chewing, to penetrate relatively deep into the food particle before it breaks apart into smaller particles. By doing so, the tooth surface is mechanically abraded by the food particle for a longer period of time than would be possible with a hard, readily breakable food particle. The disadvantage to this approach to companion animal dental hygiene is that only physical accumulation such as tartar, and perhaps some plaque structure, are removed. There is little offered in the way of a truly therapeutic or preventative effect.
These animal chews may in certain instances contain therapeutic compositions in addition to the chew itself. Compositions that have been incorporated into the animal chew may be identified as belonging to one of two categories. The first category is that of enzymatic compositions exemplified by Montgomery U.S. Pat. No. 5,310,541. Enzymatic compositions have been found to have limited antimicrobial effect. The second category is that of non-enzymatic compositions associated with the animal chews.
For example, Stacey, U.S. Pat. No. 5,296,217, described the use of a hexametaphosphate salt added to a consumable animal treat or foodstuff in order to prevent tartar accumulation in domestic animals. Not only is the residence time of the salt too short to have a significant therapeutic effect in the oral cavity of the animal, but furthermore, the hexametaphosphate is not antimicrobial. In Spanier et al., U.S. Pat. Nos. 5,114,704 and 5,011,679, a rawhide carrier coated with an inorganic pyrophosphate compound was described for purposes of preventing tartar accumulation in dogs. Pyrophosphate is a calcium chelator that limits the accumulation of tartar and reduces the tartar build-up that has occurred. However, this composition lacks antimicrobial properties.
There are a number of cationic antimicrobial agents that have been utilized in toothpastes for human dental care. These agents strongly associate with protein and are not readily released from proteinaceous substrates.
There is an unmet need for an animal chew that has effective antimicrobial properties in the oral cavity of an animal.
The invention satisfies the above need. A novel animal oral care composition is provided that includes a carrier having a negatively-charged surface and an effective dose of a therapeutic composition for achieving antimicrobial activity in the oral cavity of the animal, wherein the therapeutic composition contains at least a cationic antimicrobial substance and is in a saliva soluble form positioned close to or at the surface of the proteinaceous carrier. A method is further provided for providing dental health in an animal, including obtaining an animal oral care composition as described above and administering the composition to the animal in a form that will be voluntarily chewed by the animal.
The invention provides a composition and method directed to the dental health and well-being of animals and includes the following features. Firstly, the composition is safe for consumption, much as a foodstuff or animal feed would be; secondly, human intervention is not required in the companion animal oral care process; thirdly, in order to satisfy the self-administration rule, the composition in association with a carrier, is sufficiently palatable for the animal to maintain its interest in consuming the material; fourthly, the residence time of the composition and the carrier is sufficient to permit the desired therapeutic effect; and fifthly the therapeutic substance is readily released into saliva upon being chewed.
The present invention has accomplished all these criteria in a novel formulation. According to the invention, a carrier is utilized that has a negative surface charge. The carrier may be formed from natural or synthetic substances, and further may be inherently negatively charged, or may be coated by a reagent that imparts the negative charge to the surface of the carrier. The carrier should persist in the oral cavity of the animal for a minimum residence time of at least 1 minute. Furthermore, the carrier itself should be palatable for the animal such that the interest of the animal in the carrier is maintained.
The carrier provides a means for introducing the therapeutic agent into the oral cavity. The therapeutic substance is located on or near the surface of the chewable object in order for intimate contact between the moisture of saliva and the therapeutic substance to occur almost immediately upon the start of the chewing cycle so as to minimize the possibility that the therapeutic substance will be consumed with the chew; rather than being released in to the oral cavity.
An embodiment of the invention describes a formulation that utilizes a proteinaceous animal chew such as rawhide, and a dentally therapeutic cation, (in this example, chlorhexidine) that is maintained on the surface of the chew on the basis of charge attraction. The cationic antimicrobials become strongly bound to negatively-charged surfaces containing negatively charged moieties such as carboxylic, phosphate and sulfate moieties by forming salt bridges. Cationic antimicrobials that are released from the carrier in the presence of saliva are observed to have a long duration of action, due to their retention and adherence to the negatively-charged surface in the oral cavity, e.g., enamel hydroxyapatite, acquired pellicle protein, and the oral mucosa.
In a preferred embodiment, the cation is rapidly solubilized in the saliva of the oral cavity when the cation is combined with or deposited on the chew in the presence of an alkali metal salt such as sodium gluconate. It has unexpectedly been found that the presence of the alkali metal salt effectively prevents the cationic compound from precipitating or otherwise adhering to the proteinaceous carrier, thus rendering it readily soluble in saliva during the chewing cycle. The invention is not limited to a solubilization process that utilizes an alkali metal salt. Alternative secondary agents are contemplated that provide a means to readily release cations into saliva. Thus, the therapeutic cation is released into the salivary solution during the chewing cycle, rather than carried into the intestinal tract as a result of being bound irreversibly to the carrier.
Cationic antimicrobials contemplated to have utility in the invention include chlorhexidine diacetate, chlorhexidine digluconate, cetylpyridinium chloride, domiphen bromide, benzalkonium chloride, benzethonium chloride, and alexidene.
Alkali metal salts having utility in the invention include sodium and potassium salts of hydrochloric acid, hydrobromic acid, gluconic acid, and acetic acid.
Auxiliary ingredients such as flavorants and film-forming agents may be included in the compositions to provide a specific palatability or coating effect respectively. In particular, film formers such as hydroxypropylcellulose, carrageenan and polyvinylpyrrolidone may provide for a more uniform coating of the carrier surface with the inventive compositions. The auxiliary ingredients are not essential for the practice of the invention.
An example of the inventive composition is prepared as follows:

Proteinaceous animal chew with dentally therapeutic cation


Nuclear matrix proteins (NMP) which are characterized by a defined expression in tissue, particularly prostate tissue, are provided. These NMPs are useful markers in diagnosing and monitoring the stage of malignancy of a cell and treating cell proliferative disorders associated with the NMP. Also provided are substantially purified polypeptides and nucleotide sequences encoding the NMPs of the invention.

The present invention relates generally to nuclear matrix proteins, called xe2x80x9cNMPsxe2x80x9d here, and more specifically to novel nuclear matrix proteins of the prostate which are associated with cell-proliferative disorders.
The early diagnosis of prostate cancer is central to the effective treatment of the disease. In addition, the ability to differentiate disease with metastatic ability from prostate cancer that is not able to metastasize is important. Nuclear structural alterations are so prevalent in cancer cells that they are commonly used as a pathological marker of transformation for many types of cancer. Nuclear shape is determined in part by the nuclear matrix, the dynamic skeleton of the nucleus.
The nuclear matrix is the structural component of the nucleus that determines nuclear morphology, organizes the DNA in a three-dimensional fashion which is tissue specific, and has a central role in the regulation of a number of nuclear processes including the regulation of gene expression. The nuclear matrix has been demonstrated to play a central role in the regulation of important cellular processes such as DNA replication and transcription. Getzenberg, J. Cell Biochem. 55: 22-31 (1994). The nuclear matrix is the framework or scaffolding of the nucleus and consists of the peripheral laminas and pore complexes, an internal ribonucleic protein network, and residual nucleoli. Berezney et al., Biochem. Biophys. Res. Comm. 60: 141017 (1974). The nuclear matrix consists of approximately 10% of the nuclear proteins and is virtually devoid of lipids, DNA and histones. Fey et al., Critical Reviews in Eukaryotic Gene Expression 1: 127-44 (1991).
A majority of the known NMPs are common to all cell types and physiologic states. A number of laboratories have identified NMPs which may be unique to certain cell types or states. Mitogenic stimulation and the induction of differentiation have been demonstrated to alter the composition of nuclear matrix proteins and structure. The nuclear matrix contains a number of associated proteins that have been demonstrated to be involved in transformation. Berezney first showed that the nuclear matrix is altered in transformation, examining hepatoma nuclear matrix proteins. Berezney et al., Cancer Res. 39: 3031-39 (1979). Fey and Penman demonstrated that tumor promoters induce a specific morphologic signature in the nuclear matrix-intermediate filament scaffold of kidney cells. Fey et al., Proc. Nat""l Acad. Sci. USA 81: 859-66 (1984). Fey and Penman went on to demonstrate that the pattern of NMPs differed between normal and tumorigenic cell lines. Fey et al., loc. cit. 85: 121-25 (1989). An antibody to a nuclear matrix protein, termed NM-200.4, was raised from the breast carcinoma cell line T47D. Weidner et al., Am. J. Path. 138: 1293-98 (1991). This antibody reacts strongly with human breast carcinoma specimens as well as specimens from lung, thyroid, and ovarian cancers, but does not react with normal epithelial cells of similar origin, raising the possibility of the use of certain anti-NMP antibodies as diagnostic tools.
U.S. Pat. No. 5,824,490 discloses certain nuclear matrix proteins associated with prostate tissue, including one denoted xe2x80x9cPC-1xe2x80x9d used to identify prostate cancer. When human prostate samples were examined, nuclear matrix proteins were identified that (1) were present only in the normal prostate and were missing in both prostate cancer and benign prostatic hyperplasia (BPR) (normal pattern), (2) were found only in the prostate cancer cells and missing in the normal prostate and BPH (prostate cancer pattern), and (3) were found in both normal and BPH samples but were absent from prostate cancers. See also Getzenberg et al., Cancer Res., 1991(51):6514-20.
Additional nuclear matrix proteins associated with prostate tissue are disclosed in Partin et al., Cancer Res. 1993(53): 744-746.
The present invention relates to novel nuclear matrix proteins that are able to differentiate cancerous cells from normal cells and metastatic cancer cells from non-metastatic disease, polynucleotide sequences encoding them, polynucleotide sequences hybridizing to the sequences encoding them, antibodies directed against them, and their methods of use. In particular, these proteins are useful for diagnosing and producing treatments for cell proliferative disorders of the prostate.
According to one aspect, the present invention comprehends a purified nuclear matrix protein (NMP) or a fragment thereof, which is absent in normal prostate cells but present in cancerous prostate cells. Preferably, the protein of this embodiment is selected from the those, as described below, that are designated xe2x80x9cD-1,xe2x80x9d xe2x80x9cD-2,xe2x80x9d and xe2x80x9cD-3.xe2x80x9d The proteins D-1, D-2, and D-3 are present in both metastatic and non-metastatic cancerous prostate cells.
According to another aspect, the present invention is directed to a purified NMP or a fragment thereof, which is present in normal prostate cells but absent in cancerous prostate cells. Preferably, the protein of this embodiment is selected from the proteins described below which have been designated NDP-1, NDP-2, NDP-3, NDP-4, NDP-5, NDP6, NDP-7, NDP-8, NDP-9, and NDP-10.
According to another aspect, the present invention is directed generally to a method for differentiating a metastatic cell (preferably, prostate) from a non-metastatic cell (preferably prostate) comprising determining the presence or absence of a nuclear matrix protein unique to a metastatic cell or a non-metastatic cell. In particular, nuclear matrix proteins of the prostate are disclosed herein which are capable of differentiating between a metastatic cancer cell and non-metastatic cell of the prostate.
According to another aspect, the present invention is directed to a purified NMP or a fragment thereof, which is present in metastatic but absent in non-metastatic prostate tumors and absent in normal prostate cells. Preferably, the protein of this embodiment is selected from the proteins described below which have been designated as AM-1 and AM-2.
According to another aspect, the present invention is directed to a purified NMP or a fragment thereof, which is absent in metastatic but present in non-metastatic prostate tumors. Preferably, the protein of this embodiment is selected from the proteins described below which have been designated as G-1 and G-2. These two proteins, G-1 and G-2, are also not present in normal prostate cells.
Preferably, the fragments of the NMPs in the above embodiments are immunogenic fragments. It also is preferable that, in the embodiments discussed above, the NMPs are derived from humans.
Unless otherwise specified, the terms xe2x80x9caxe2x80x9d, xe2x80x9canxe2x80x9d or xe2x80x9cthexe2x80x9d mean one or more.
The phrase xe2x80x9cpurified nuclear matrix proteinxe2x80x9d means a protein of the nuclear matrix which has been separated from at least one cellular component. The phrase covers both purified nuclear matrix proteins produced recombinantly and those produced by extraction from a natural source.
Another embodiment of the present invention is a purified polynucleotide sequence encoding the above-identified NMPs or NMP fragments of the preceding embodiments. Another embodiment is a purified polynucleotide sequence which hybridizes to the polynucleotide sequence encoding the above-mentioned NMPs or NMP fragments.
Another embodiment is a host cell transformed with a polynucleotide sequence encoding the above-mentioned NMPs or NMP fragments. Transformation of a host cell with recombinant DNA may be carried out by conventional techniques known in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after the exponential growth phase and subsequently treated by the CaC12 method by procedures well known in the art. Alternatively, MgC12 or RbC1 can be used. Transformation can also be performed after forming a protoplast of the host cell or by electroporation.
When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be cotransformed with DNA sequences encoding the NMPs of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (5V40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein. EUKARYOTIC VIRAL VECTORS Gluzman (ed.), Cold Spring Harbor Laboratory, 1982.
Isolation and purification of the NMPs or NMP fragments expressed by a transformed host may be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies. Antibodies provided in the present invention are immuno-reactive with NMP polypeptide or fragments thereof.
Another embodiment is a recombinant expression vector containing the above-mentioned polynucleotide sequences. Preferably, the vector is a virus. Preferred viruses are RNA viruses and preferred RNA viruses are retroviruses. Another preferred vector is a liposome, preferably a target-specific liposome which may be targeted with, for example, an antibody or ligand. Another preferred vector is a plasmid.
Another embodiment is an antibody which binds to the above-mentioned NMPs or NMP fragments. The antibody may be polyclonal or monoclonal. Using the NMPs isolated by the inventors, antibodies have been prepared which are capable of differentiating between cancerous prostate tissue and normal prostate tissue in both the Dunning model and in human tissues. One embodiment of the present invention is directed to these antibodies. Furthermore, another embodiment of the present invention is directed to antibodies which can detect individuals with prostate cancer that has the ability to advance and/or metastasize.
Another embodiment is a method for detecting a cell proliferative disorder in a subject or for detecting individuals at risk of developing a cell proliferative disorder, preferably prostate cancer, comprising contacting a cellular component from the subject with an antibody or nucleic acid probe which binds to a cellular component associated with the cell proliferative disorder. More preferably, the method is a method for differentiating between prostate cancer that has the potential to metastasize and prostate cancer that lacks the potential to metastasize. Preferably, the cellular component is taken from the subject""s prostate and is preferably a nucleic acid. Preferably, the nucleic acid is DNA encoding the above-mentioned NMPs or NMP fragments. Also preferred as a nucleic acid is RNA. Another preferred cellular component is the above-mentioned NMPs or NMP fragments. The differentiation method may be practiced by detecting a single NMP and/or its corresponding DNA or a combination of one or more of the above NMPs and/or their DNA.
Preferably, the nucleic acid probe specifically hybridizes to the above-mentioned cellular component. When the reagent is a nucleic acid probe, it is preferably detectably labeled. Preferred labels include a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, and an enzyme.
Alternatively, if the cellular component is an NMP or NMP fragment, then an antibody is used which specifically binds to the NMP or NMP fragment. As noted above, the antibody may be monoclonal or polyclonal.
Another embodiment is a method of treating a cell proliferative disorder associated with a protein selected from the group consisting of D-1, D-2, D-3, AM-1, AM-2, NDP-1, NDP-2, NDP-3, NDP4, NDP-5, NDP-6, NDP-7, NDP-8, NDP-9, NDP-10, G-1, and G-2, comprising administering to a subject with the disorder a therapeutically effective amount of an antisense polynucleotide sequence that blocks the sequences encoding the above-mentioned NMPs. In this embodiment, the treatment is designed to block the expression of one or more NMPs which give rise to the cell proliferative disorder. More preferably, the method is a method of inhibiting metastasis of a cell proliferative disorder and preferably the disorder is prostate cancer.
In an alternative method of treatment, instead of using an antisense polynucleotide sequence, a polynucleotide sequence is used which encodes one of the above-mentioned NMPs. In this embodiment, the treatment is designed to provide the subject with one or more NMPs that prevent or ameliorate the cell proliferative disorder.
In another method of treatment, an antibody is administered to the subject which is capable of blocking the function of one or more of the above NMPs.
Another embodiment is a method of gene therapy, comprising introducing into the cells of a host subject an expression vector comprising a polynucleotide sequence encoding one or more of the above-mentioned NMPs. Preferably, the expression vector is introduced into the cells of the host subject ex vivo, yielding transformed cells, and the transformed cells then are reintroduced into the subject. A preferred expression vector for this purpose is an RNA virus, preferably a retrovirus.
Another embodiment of the present invention relates to a method for identifying a composition which blocks or enhances the function of a prostate cell NMP. The inventive method comprises: (a) incubating NMP-containing prostate cells with a test composition under conditions that allow the prostate cells and test composition to interact, and (b) measuring whether the test composition blocks or enhances the function of the prostate cell NMP.
Another embodiment of the present invention is a kit for detecting a cell-proliferative disorder of the prostate comprising a nucleic acid probe that binds to a polynucleotide sequence encoding one of the above-mentioned NMPs. Preferably, the probe is labeled for ease of detection with a label as described above. Alternatively, the kit may comprise an antibody which specifically binds to one of the above-mentioned NMPs. Still another alternative is to use an oligonucleotide primer in the kit that permits amplification of a target polynucleotide sequence encoding one of the above-mentioned NMPs, for example, by polymerase chain reaction (PCR) amplification.
The NMPs of the invention include fragments, and conservatively substituted variants thereof. Minor modifications of the NMP primary amino acid sequence may result in proteins which have substantially equivalent activity as compared to the NMP polypeptide described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous, and can include deletion of non-essential amino acids. All of the polypeptides produced by these modifications are included herein as long as the biological activity of the native NMP still exists. Further, deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its biological activity. This can lead to the development of a smaller active molecule which would have broader utility.
The term xe2x80x9cconservative substitutionxe2x80x9d as used herein denotes the replacement of an amino acid residue by a structurally similar residue. Examples of conservative substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like.
Peptides of the invention can be synthesized by the well known solid phase peptide synthesis methods described, for example, by Merrifield, J. Am. Chem. Soc. 85: 2149 (1962), and by Stewart and Young, SOLID PHASE PEPTIDES SYNTHESIS 27-62 (Freeman Publ., 1969).
The polyclonal and monoclonal antibodies of the invention are immunoreactive with the NMPs or immunogenic fragments of the NMPs. If desired, polyclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which NMP polypeptide is bound or by utilizing common nuclear matrix proteins to selectively remove non-specific antibodies. Antibodies which consist essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided. The term xe2x80x9cantibodyxe2x80x9d as used in this invention includes intact molecules as well as fragments thereof, such as Fab and F(abxe2x80x2)2 fragments, which are functionally capable of binding an epitopic determinant of an NMP.
A preferred method for the identification and isolation of antibody binding domains which exhibit binding with NMP is the bacteriophage X vector system. This vector system has been used to express a combinatorial library of Fab fragments from the mouse antibody repertoire in Escherichia coli, see Huse et al., Science 246: 1275-81 (1989), and from the human antibody repertoire. Nullinax et al., Proc. Nat""l Acad. Sci. USA 87: 8095-99 (1990).
As used herein, the term xe2x80x9ccell-proliferative disorderxe2x80x9d denotes malignant as well as non-malignant (or benign) disorders of the prostate. This term further encompasses hyperplastic disorders of the prostate. The cells comprising these proliferative disorders often appear morphologically and genotypically to differ from the surrounding normal tissue. As noted above, cell- proliferative disorders may be associated, for example, with expression or absence of expression of the NMPs of the invention. Expression of an NMP at an inappropriate time during the cell cycle or in an incorrect cell type may result in a cell-proliferative disorder. The NMP- encoding polynucleotide in the form of an antisense polynucleotide is useful in treating hyperplasia and malignancies of the prostate. When the cell-proliferative disorder is associated with NMP expression, an antisense NMP polynucleotide sequence or NMP binding antibody can be introduced into the prostate cells to block the expression and/or function of the NNP. Alternatively, when the cell proliferative disorder is associated with under-expression or expression of a mutant NMP polypeptide, a polynucleotide sequence encoding the missing or under-expressed NMP can be introduced into the cell.
For purposes of the invention, an antibody or nucleic acid probe specific for an NMP may be used to detect the presence of the NMP polypeptide (in the case of an antibody probe) or polynucleotide (in the case of the nucleic acid probe) in biological fluids or tissues suspected of containing the NMP. Oligonucleotide primers based on any coding sequence region in the NMP sequence are useful for amplifying DNA or RNA, for example by PCR. Any specimen containing a detectable amount of antigen can be used. A preferred sample of this invention is tissue taken from the prostate. Alternatively, biological fluids which may contain cells of the prostate may be used.
The term xe2x80x9csubjectxe2x80x9d as used herein refers to mammals, preferably humans.
Another technique which may also result in greater sensitivity consists of coupling the probe to low molecular weight haptens. These haptens can then be specifically detected by means of a second reaction. For example, it is common to use such haptens as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, and fluorescein, which can react with specific antihapten antibodies.
The method for detecting a cell expressing a particular NMP of the invention or a cell-proliferative disorder associated with an NMP, described above, can be utilized for detection of residual prostate cancer or other malignancies or benign hyperplasia conditions in a subject in a state of clinical remission. Additionally, the method for detecting NMP polypeptide in cells is useful for detecting a cell-proliferative disorder by identifying cells expressing specific NMPs in comparison with NMPs expressed in normal cells. Using the method of the invention, NMP expression can be identified in a cell and the appropriate course of treatment can be employed (e.g., NMP-encoding or antisense gene therapy, as well as conventional chemotherapy). Since the expression pattern of the NMPs of the invention vary with the stage of malignancy of a cell, a sample of prostate tissue can be screened with a panel of NMP-specific reagents, e.g., nucleic acid probes or antibodies to NMPs, to detect NMP expression and diagnose the stage of malignancy of the cell.
The monoclonal antibodies of the invention are suitable for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. In addition, the monoclonal antibodies in these immunoassays can be detectably labeled in various ways. Examples of types of immunoassays which can utilize monoclonal antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay. Detection of the antigens using the monoclonal antibodies of the invention can be performed utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Alternatively, the antibody of the invention can be used to detect NMPs present in electrophoretically dispersed gel protocols such as Western blots and two-dimensional gels.
The monoclonal antibodies of the invention can be bound to many different carriers and used to detect the presence of NMP. Examples of wellxe2x80x94known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the invention.
In performing the assays it may be desirable to include certain xe2x80x9cblockersxe2x80x9d in the incubation medium (usually added with the labeled soluble antibody). The xe2x80x9cblockersxe2x80x9d are added to assure that non-specific proteins, proteases, oranti-heterophilic immunoglobulins to anti-NMP immunoglobulins present in the experimental sample do not cross-link or destroy the antibodies on the solid phase support, or the radiolabeled indicator antibody, to yield false positive or false negative results. The selection of xe2x80x9cblockersxe2x80x9d therefore may add substantially to the specificity of the assays described in the present invention.
It has been found that a number of nonrelevant (i.e., nonspecific) antibodies of the same class or subclass (isotype) as those used in the assays (e.g., IgGi, IgG2a, 1gM, etc.) can be used as xe2x80x9cblockers. xe2x80x9d The xe2x80x9cblockersxe2x80x9d are used at a level high enough to maintain the proper sensitivity yet inhibit any unwanted interference by mutually occurring cross reactive proteins in the specimen (normally 1-100 xcexcg/xcexcl).
In this description, the term xe2x80x9cepitopexe2x80x9d denotes any determinant capable of specific interaction with the monoclonal antibodies of the invention. Epitopic determinants usually comprise chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
In using the monoclonal antibodies of the invention for the in vivo detection of antigen, the detectably labeled monoclonal antibody is given in a dose which is diagnostically effective. The term xe2x80x9cdiagnostically effectivexe2x80x9d means that the amount of delectably labeled monoclonal antibody is administered in sufficient quantity to enable detection of the site having the NMP antigen for which the monoclonal antibody is specific.
The dosage of detectably labeled monoclonal antibody for in vivo diagnosis will vary depending on such factors as age, sex, and extent of disease of the individual. The dosage of monoclonal antibody can vary from about 0.001 mg/in2, to about 500 mg/in2, preferably 0.1 mg/in2 to about 200 mg/in2, most preferably about 0.1 mg/in2 to about 10 mg/rn2. Such dosages may vary, for example, depending on whether multiple injections are given, tumor burden, and other factors.
For in vivo diagnostic imaging, the type of detection instrument available is a major factor in selecting a given radioisotope. The radioisotope chosen must have a type of decay which is detectable for a given type of instrument. Still another important factor in selecting a radioisotope for in vivo diagnosis is that the half-life of the radioisotope be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation with respect to the host is minimized. Ideally, a radioisotope used for in vivo imaging will lack a particle emission, but produce a large number of photons in the 140-250 keV range, which may be readily detected by conventional gamma cameras.
For in vivo diagnosis, radioisotopes may be bound to immunoglobulin either directly or indirectly by using an intermediate functional group. Intermediate functional groups which often are used to bind radioisotopes which exist as metallic ions to immunoglobulins are the bifunctional chelating agents such as diethylene-triaminepentacetic acid (DTPA) and ethylenediarninetetraacetic acid (EDTA) and similar molecules. Typical examples of radioisotopes which can be bound to the monoclonal antibodies of the invention are 111In, 97Ru, 67Ga, Ga, 72As, 89Zr, and 201T1.
The monoclonal antibodies of the invention can also be labeled with a paramagnetic isotope for purposes of in vivo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR). In general, any conventional method for visualizing diagnostic imaging can be utilized. Usually gamma and positron emitting radioisotopes are used for camera imaging and para-magnetic isotopes for MRI. Elements which are particularly useful in such techniques include 57Gd, 55Mn, 162Dy, 52Cr, and 56Fe.
The monoclonal antibodies of the invention can be used to monitor the course of amelioration of an NMP-associated cell-proliferative disorder. Thus, by measuring the increase or decrease in the number of cells expressing a NMP or changes in NMP present in various body fluids, such as ejaculate or urine, it would be possible to determine whether a particular therapeutic regimen aimed at ameliorating the disorder is effective.
The monoclonal antibodies of the invention can also be used, alone or in combination with effector cells, see Douillard et al., Hybridoma 5 (Supp. 1): S139 (1986), for immunotherapy in an animal having a cell proliferative disorder which expresses NMP polypeptide with epitopes reactive with the monoclonal antibodies of the invention.
When used for immunotherapy, the monoclonal antibodies of the invention may be unlabeled or attached to a therapeutic agent. These agents can be coupled either directly or indirectly to the monoclonal antibodies of the invention. One example of indirect coupling is by use of a spacer moiety. These spacer moieties, in turn, can be either insoluble or soluble, see Diener et al., Science 231: 148 (1986), and can be selected to enable drug release from the monoclonal antibody molecule at the target site. Examples of therapeutic agents which can be coupled to the monoclonal antibodies of the invention for immunotherapy are drugs, radioisotopes, lectins, and toxins.
The drugs which can be conjugated to the monoclonal antibodies of the invention include non-proteinaceous as well as proteinaceous drugs. The terms xe2x80x9cnon-proteinaceous drugsxe2x80x9d encompasses compounds which are classically referred to as drugs, for example, mitomycin C, daunorubicin, vinblastine, and others used to treat cancer.
The proteinaceous drugs with which the monoclonal antibodies of the invention can be joined include immunomodulators and other biological response modifiers. The term xe2x80x9cbiological response modifiersxe2x80x9d encompasses substances which are involved in modifying the immune response in such manner as to enhance the destruction of an NMP-associated tumor for which the monoclonal antibodies of the invention are specific. Examples of immune response modifiers include such compounds as lymphokines. Lymphokines include tumor necrosis factor, the interleukins, lymphotoxin, macrophage activating factor, migration inhibition factor, colony stimulating factor, and interferon. Interferons with which the monoclonal antibodies of the invention can be labeled include alpha-interferon, beta-interferon and gamma-interferon and their subtypes.
In using radioisotopically conjugated monoclonal antibodies of the invention for immunotherapy, certain isotopes may be more preferable than others depending on such factors as tumor cell distribution as well as isotope stability and emission. If desired, the tumor cell distribution can be evaluated by the in vivo diagnostic techniques described above. Depending on the cell proliferative disease some emitters may be preferable to others. In general, alpha and beta particle-emitting radioisotopes are preferred in immunotherapy. For example, if an animal has solid tumor foci a high energy beta emitter capable of penetrating several millimeters of tissue, such as 9%, may be preferable. On the other hand, if the cell proliferative disorder consists of simple target cells, as in the case of leukemia, a short range, high energy alpha emitter, such as Bi, may be preferable. Examples of radioisotopes which can be bound to the monoclonal antibodies of the invention for therapeutic purposes are radioisotopes of I, Y, Cu, Bi, At, Pb, Sc, Pd, Zn, and Re.
Lectins are proteins, usually isolated from plant material, which bind to specific sugar moieties. Many lectins are also able to agglutinate cells and stimulate lymphocytes. Ricin is a toxic lectin which has been used immunotherapeutically. The alpha-peptide chain of ricin, which is responsible for toxicity, may be bound to the antibody of the invention to enable site specific delivery of the toxic effect.
Toxins are poisonous substances produced by plants, animals, or microorganisms, that, in sufficient dose, are often lethal. Diphtheria toxin is a substance produced by Corynebacterium diphtheria which can be used therapeutically. This toxin consists of an alpha and beta subunit which under proper conditions can be separated. The toxic A component can be bound to an antibody and used for site specific delivery to a NMP bearing cell.
The monoclonal antibodies of the invention can be used in combination with alpha-interferon. This treatment modality enhances monoclonal antibody targeting of carcinomas by increasing the expression of monoclonal antibody reactive antigen by the carcinoma cells. Greiner et al., Science 235: 895 (1987). Alternatively, the monoclonal antibody of the invention can be used, for example, in combination with gamma-interferon to thereby activate and increase the expression of Fc receptors by effector cells which, in turn, results in an enhanced binding of the monoclonal antibody to the effector cell and killing of target tumor cells.
It also is possible to utilize liposomes with the monoclonal antibodies of the invention in their membrane to specifically deliver the liposome to the tumor expressing NMP. These liposomes can be produced such that they contain, in addition to the monoclonal antibody, such immunotherapeutic agents as those described above which would then be released at the tumor site. Wolff et al., Biochemical et Biophysical Acta 802: 259 (1984).
The dosage ranges for the administration of monoclonal antibodies of the invention are those large enough to produce the desired effect in which the symptoms of the malignant disease are ameliorated. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication. Dosage can vary from about 0.1 mg/kg to about 2000 mg/kg, preferably about 0.1 mg/kg to about 500 mg/kg, in one or more dose administrations daily, for one or several days. Generally, when the monoclonal antibodies of the invention are administered conjugated with therapeutic agents, lower dosages, comparable to those used for in vivo diagnostic imaging, can be used.
The monoclonal antibodies of the invention can be administered parenterally by injection or by gradual perfusion over time. The monoclonal antibodies of the invention can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally, alone or in combination with effector cells.
The present invention also provides a method for treating a subject with an NMP-associated cell-proliferative disorder using an NMP nucleotide sequence. An NMP nucleotide sequence which may encode a suppressor polypeptide may be under-expressed as compared to expression in a normal cell, therefore it is possible to design appropriate therapeutic or diagnostic techniques directed to this sequence. Thus, where a cell-proliferative disorder is associated with the expression of an NMP associated with malignancy, nucleic acid sequences that interfere with NMP expression at the translational level can be used. This approach utilizes, for example, antisense nucleic acid and ribozymes to block translation of a specific NMP mRNA, either by masking that mRNA with an antisense nucleic acid or by cleaving it with a ribozyme. In cases when a cell proliferative disorder or abnormal cell phenotype is associated with the under expression of NMP suppressor for example, nucleic acid sequences encoding UMP (sense) could be administered to the subject with the disorder.
Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule. Weintaub, Scientific American 262: 40 (1990). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA since the cell will not translate a mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to be expressed than larger molecules when introduced into the target NMP-producing dell.
Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it. Cech, J. Amer. Med. Assn. 260:3030 (1988). A major advantage of this approach is that, because they are sequence-specific, only in RNAs with particular sequences are inactivated.
There are two basic types of ribozymes namely, tetrahymena-type (Hasselhoff, Nature 334:585 (1988)) and xe2x80x9chammerheadxe2x80x9d-type. Tetrahymena-type ribozymes recognize sequences which are four bases in length, while xe2x80x9chammerheadxe2x80x9d-type ribozymes recognize base sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that that sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating a specific mRNA species and 18-based recognition sequences are preferable to shorter recognition sequences.
The present invention also provides gene therapy for the treatment of cell proliferative disorders which are mediated by NMP. Such therapy requires introduction of the appropriate NMP polynucleotide sequence (antisense or encoding strand) into cells of subjects having the proliferative disorder. Delivery of antisense NMP polynucleotides can be achieved using a recombinant expression vector such as a chimeric virus or a liposome. Disorders associated with under-expression of an NMP or expression of a cancer-associated NMP can be treated using gene therapy with the encoding or antisense nucleotide sequences, respectively.
Various viral vectors which can be utilized for gene therapy as taught herein include adenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such as a retrovirus. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), inurine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV) A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. By inserting an NMP sequence of interest into the viral vector along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is rendered target specific. Retroviral vectors can be made target specific by inserting, for example, a polynucleotide encoding a sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using an antibody to target the retroviral vector.
Since recombinant retroviruses are defective in one or more genes, they require assistance in order to produce infectious vector particles. Helper cell lines which have deletions of the packaging signal include but are not limited to W2, PA317 and PA12, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced.
Alternatively, NIH 3T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.
Other targeted delivery systems for NMP antisense polynucleotides include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (ULV), which range in size from 0.2-4.0 pm can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form. Fraley et al., Trends Biochem. Sci. 6: 77 (1981).
The composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.
Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidyiserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
The targeting of liposomes has been classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
The surface of the targeted delivery system may be modified in a variety of ways. In the case of a hyposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer. Various linking groups can be used for joining the lipid chains to the targeting ligand.
In general, the compounds bound to the surface of the targeted delivery system will be ligands and receptors which will allow the targeted delivery system to find and xe2x80x9chome inxe2x80x9d on the desired cells. A ligand may be any compound of interest which will bind to another compound, such as a receptor.
In general, surface membrane proteins which bind to specific effector molecules are referred to as receptors. In the present invention, antibodies of the invention are preferred receptors. Antibodies can be used to target liposomes to specific cell-surface ligands, in this case the NMPs of choice. Preferably, the target tissue is prostate tissue. A number of procedures can be used to covalently attach either polyclonal or monoclonal antibodies to a liposome bilayer. Antibody-targeted liposomes can include monoclonal or polygonal antibodies or fragments thereof such as Fab, or F(abxe2x80x2)2, as long as they bind efficiently to an the antigenic epitope on the target cells.
Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer""s dextrose, dextrose and sodium chloride, lactated Ringer""s intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such an those based on Ringer""s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents and inert gases and the like.
The invention also relates to a method for preparing a medicament or pharmaceutical composition comprising the polynucleotides or the monoclonal antibodies of the invention, the medicament being used for therapy of NMP-associated cell proliferative disorders.

Nuclear matrix proteins polynucleotide sequences encoding them and their use