Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
  • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
  • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
  • C07H13/06—Fatty acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0043—Nose
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant

Definitions

• the compounds monophosphoryl lipid A (MLA) and 3-O-deacylated monophosphoryl lipid A (3D-MLA) are attenuated derivatives of the lipid A component of bacterial lipopolysaccharide (LPS).
• LPS and lipid A are potent immunostimulants inducing both a humoral antibody response and a cell-mediated immune response in patients administered the compounds.
• Lipid A and LPS however can also display toxic side-effects such as pyrogenicity and local Shwarzman reactions.
• MLA and 3D-MLA are lipid A-like molecules that have been modified to attenuate the toxicity of LPS.
• the MLA and 3D-MLA molecules have a sugar backbone onto which long chain fatty acids are attached.
• the backbone is comprised of two six carbon sugar rings in glycosidic linkage.
• MLA and 3D-MLA are phosphorylated at the 4 position.
• Five to eight long chain fatty acids (12-14 carbons) are attached to the sugar backbone making MLA and 3D-MLA very hydrophobic molecules which are not readily water soluble.
• the attenuated lipid A derivatives (ALDs) MLA and 3D-MLA are used as immunologic adjuvants in prophylactic vaccines for infectious disease and therapeutic vaccines for the treatment of cancerous tumors and chronic infections.
• Antigen preparations included in most vaccines are often complicated mixtures of water-soluble proteins making it difficult to formulate the water insoluble adjuvant in a water based vaccine. Therefore, MLA and 3D-MLA must be first mixed with solvents before they are added to the antigen preparation. However, the presence of solvents can further complicate the formulation of the vaccine, and in some cases can reduce the efficiency of its components. Further, solvents can irritate mucosal surfaces or cause inflammation at an injection site.
• a simple formulation of MLA or 3D-MLA containing no interfering co-solvents would allow maximum benefits to be derived from both the adjuvant and the antigen in a vaccine composition. The instant invention satisfies this need.
• a derivative A derivative (ALD) and a surfactant and methods for its preparation and storage.
• Attenuated lipid A derivatives useful according to the subject invention include monophosphoryl lipid A (MLA) and 3-O-deacylated monophosphoryl lipid A (3D- MLA).
• MLA monophosphoryl lipid A
• 3D- MLA 3-O-deacylated monophosphoryl lipid A
• Aqueous formulations of MLA (MLA/AF) or 3D-MLA (3D-MLA/AF) eliminate the need for undesirable solvents or a co-solvent system for vaccine preparation.
• the invention provides a stable aqueous composition of the ALD and a surfactant which when administered to mice with an antigen, enhances the cellular and humoral immune response of the animal to that antigen.
• the aqueous formulation of the present invention induces high levels of serum and mucosal secreted IgA in immunized animals when administered intranasally.
• An embodiment of the claimed aqueous composition comprises a MLA or 3D-MLAto surfactant molar ratio of about 4:1 and has a particle size of approximately 50-70 nm.
• l,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) is a preferred surfactant.
• DPPC l,2-Dipalmitoyl-sn-glycero-3-phosphocholine
• glycerol is added to the subject aqueous formulation before lyophilization, the composition is restored upon reconstitution with no additional sonication. Successful storage of the subject composition in a lyophilized state allows for convenient storage and transport of the aqueous formulation or vaccine compositions comprising the formulation.
• a method of preparing the aqueous composition is disclosed.
• the ALD and the surfactant are dissolved and uniformly admixed in ethanol.
• the ethanol is then evaporated leaving a film.
• Water is added to the film.
• the ALD and surfactant are suspended in the water by sonication.
• the suspension is sonicated until clear.
• Animals administered the claimed composition with an antigen display enhanced humoral and cellular immune responses to that antigen. Methods for using the composition to enhance these responses are also disclosed and claimed.
• Figure 1 a-d show the antibody titers of mice administered tetanus toxoid (TT) antigen in 3-O-deacylated monophosphoryl lipid A-aqueous formula (3D- MLA/AF) ir or tetanus toxoid antigen in saline o.
• Figure la shows the total IgG antibody titers of mice administered the tetanus toxoid antigen.
• Figure lb shows the IgG2a antibody titers of mice administered the tetanus toxoid antigen.
• Figure lc shows the IgG2b antibody titers of mice administered the tetanus toxoid antigen and
• Figure Id shows the IgGl antibody titers for the animals.
• Figure 2 shows the T-cell proliferative response in mice immunized with a purified protein derivative.
• the proliferative response in mice administered tetanus toxoid in 3D-MLA/AF ic and normal controlso are shown 14 days post primary vaccination.
• the subject invention involves an aqueous adjuvant formulation of an attenuated lipid A derivative (ALD).
• ALD attenuated lipid A derivative
• the ALD and a surfactant are suspended in water in a molar ratio of approximately 4: 1 and sonicated to yield a suspension having a particle size of approximately 50-70 run.
• an attenuated lipid A derivative can be formulated into an aqueous composition to provide a potent adjuvant.
• An attenuated lipid A derivative is a lipid A-like compound which displays the advantageous immunostimulatory properties of lipid A yet exhibits less of the adverse side affects of that compound.
• monophosphoryl lipid A (MLA) and 3-O-deacylated monophosphoryl lipid A (3D-MLA) are ALDs that are potent immuno stimulants but are surprisingly less toxic than lipid A. Both MLA and 3D-MLA can be used in the compositions of the subject invention and are known and need not be described in detail herein. See for example U.S. Patent No.
• monophosphoryl lipid A as used herein is derived from lipid A, a component of enterobacterial lipopolysaccharides (LPS), a potent but highly toxic immune system modulator.
• LPS enterobacterial lipopolysaccharides
• MLA monophosphoryl lipid A
• LPS enterobacterial lipopolysaccharides
• the core carbohydrate is removed from the 6 position of the non-reducing glucosamine during this treatment.
• the resulting product exhibits considerably attenuated levels of the endotoxic activities normally associated with the endotoxin starting material, such as pyrogenicity, local Shwarzman reactivity, and toxicity as evaluated in the chick embryo 50% lethal dose assay (CELD 50 ).
• CELD 50 50% lethal dose assay
• 3-O-deacylated monophosphoryl lipid A 3D-MLA
• 3D-MLA is known as set forth in U.S. patent No. 4,912,094, reexamination certificate B 1 4,912,094 (the '094 patent), and differs from MLA in that there is selectively removed from the MLA molecule the ⁇ -hydroxymyristic acyl residue that is ester linked to the reducing-end glucosamine at position 3 under conditions that do not adversely affect the other groups.
• 3-O-deacylated monophosphoryl lipid A is available from Ribi ImmunoChem Research, Inc., Hamilton, Montana 59840.
• the MLA and 3D-MLA molecules are a composite or mixture of a number of fatty acid substitution patterns, i.e., heptaacyl, hexaacyl, pentaacyl, etc., with varying fatty acid chain lengths
• these various forms of MLA and 3D-MLA are encompassed by this invention
• mixtures of forms of a compound as well as individual compounds produced by synthetic or semisynthetic means are encompassed by this invention.
• the lipid A backbone that is illustrated in the -094 patent corresponds to the product that is obtained by 3-deacylation of heptaacyl lipid A from S. minmsota R 595
• Other fatty acid substitution patterns are encompassed by this disclosure; the essential feature is that the material be 3-O- deacylated
• the modified 3D-MLA utilized in the present invention is prepared by subjecting MLA to alkaline hydrolysis under conditions that result in the loss of but a single fatty acid from position 3 of the lipid A backbone ⁇ -hydroxymyristic fatty acid at position 3 is unusually labile in alkaline media. It requires only very mild alkaline treatment to completely 3 -deacylate lipid A The other ester linkages in lipid A require somewhat stronger conditions before hydrolysis will occur so that it is possible to selectively deacylate these materials at position 3 without significantly affecting the rest of the molecule The reason for the unusual sensitivity to alkaline media of the ester-linked ⁇ -hydroxymyristic fatty acid at position 3 is not known at this time
• hydrolysis can be carried out in aqueous or organic media
• solvents include methanol (alcohols), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), chloroform, dichloromethane, and the like, as well as mixtures thereof Combinations of water and one or more of the mentioned organic solvents also can be employed.
• the alkaline base can be chosen from among various hydroxides, carbonates, phosphates and amines
• Illustrative bases include the inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, and the like, and organic bases such as alkyl amines, and include, but are not limited to, diethylarnine, triethylamine, and the like
• the pH is typically between approximately 10 and 14 with a pH of about 12 to about 13.5 being the preferred range.
• the hydrolysis reaction is typically carried out at a temperature of from about 20°C to about 80°C, preferably about 50 °C to 60 °C for a period of about 10 to about 30 minutes.
• the hydrolysis can be conducted in 3% triethylamine in water at room temperature (22° -25 °C) for a period of 48 hours.
• the only requirement in the choice of temperature and time of hydrolysis is that deacylation occurs to remove only the ⁇ -hydoxymyristic at position 3.
• the attenuated lipid A derivative (ALD) is combined with the surfactant each being dissolved in a solvent.
• the solvent is evaporated leaving a film.
• Water is added to the film and the resulting suspension is sonicated while heated until clear.
• the final suspension has a particle size of approximately 40-150 nm and preferably from about 50 to about 70 nm.
• the ALD and surfactant are combined at a molar ratio of about 10 parts ALD to from about 1 part to about 5 parts surfactant.
• the components are combined in a molar ratio of about 4 parts ALD to 1 part surfactant.
• Surfactants contemplated for use in the compositions of the subject invention are non-immunostimulatory or non-reactive displaying little or no independent biological activity.
• the terms non-immunostimulatory and non- reactive means the surfactants show little or no appreciable biological activity above non-immune controls.
• Non-immunostimulatory surfactants useful according to the subject invention include but are not limited to bile salts, natural phospholipids and sphingolipids.
• Bile salts such as glycodeoxycholate and deoxycholate are useful as surfactants in the claimed compositions.
• Other suitable surfactants include sphingolipids such as sphingomyelin and sphingosine and phospholipids such as egg phosphatidylcholine, l,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine, L- ⁇ -
• the phospholipid 1,2-Dipalmitoyl-sn- glycero-3-phosphocholine is the surfactant.
• DPPC is accepted for use in humans and is especially effective when the formulation is administered intranasally.
• the ALD and surfactant are dissolved and thoroughly admixed in a solvent.
• Aqueous or organic solvents useful according to the subject invention include chloroform, alcohols (eg. ethanol), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and the like, as well as mixtures thereof.
• alcohols eg. ethanol
• DMSO dimethyl sulfoxide
• DMF dimethylformamide
• the solvent is evaporated from the mixture of ALD and surfactant leaving a film.
• Water is added to the film and the resulting suspension is sonicated while heated until clear. It is preferred that the suspension be sonicated in a water bath sonicator.
• the water bath temperature can be from 40 °C to 80 °C and preferably about 60 °C.
• the suspension can be sonicated for periods of 5 minutes to approximately one hour until clear. Periods of sonication will vary depending upon the volume and concentration of the suspension but can be readily determined by one skilled in the art.
• the final suspension has a particle size of approximately 40- 150 nm and preferably from about 50 to about 70 nm.
• the aqueous formulation of the subject invention can be lyophilized for shipment and storage.
• the composition is lyophilized in the presence of glycerol, unexpectedly, it can be reconstituted without additional sonication.
• Glycerol present in the composition at about 2 percent to about 40 percent, and preferably about 2 to about 10 percent volume to volume, allows the lyophilized composition to be restored upon the addition of water.
• the ability to reconstitute the aqueous formulation to its original particle size by simply adding water is a distinct advantage for vaccination in the field away from laboratory equipment.
• compositions of the subject invention are believed to be protected from hydrolysis in this lyophilized state
• concentration of ALD presented to the vaccinee can be vaned by adjusting the volume for reconstitution For example, ten milliliters of a composition containing
• 1 mg/ml of ALD can be lyophilized and reconstituted with 1 ml of water to yield aqueous compositions of 10 mg/ml
• ALD Glycerol also stabilizes protein antigens present in vaccine compositions of the subject invention dunng lyophilization
• Other components which could be used to stabilize the subject composition for lyophilization include but are not limited to polypropylene glycol, polyethylene glycol or other poly alcohols appropnate for parenteral use
• composition of the subject invention is administered to a warm-blooded animal with an antigen to enhance the immune response of the animal to that antigen
• the composition of the subject invention enhances both the humoral immune response of an animal to an antigen as well as the cellular immune response
• the amount of antigen administered to elicit the desired response can be readily determined by one skilled in the art and will vary with the type of antigen administered, route of administration and immunization schedules For example, 0 1 ⁇ g of tetanus toxoid administered with the claimed composition subcutaneously to a mouse in two immunizations 21 days apart elicits a humoral immune response to that antigen Administered mtranasally, the composition of the subject invention and an antigen stimulate the production of cytotoxic T-lymphocytes Hepatitis B surface antigen (2 5 ⁇ g) administered mtranasally at days 0 and 21 in the claimed composition stimulated the production of cytotoxic T-lymphocytes in immunized animals Further, the
• mice models presented herein are representative of warm blooded animals and correlate reasonably with events for other warm blooded animals, including humans. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
• Example 1 Preparation of an Aqueous Formulation of an Attenuated Lipid A Derivative.
• An aqueous preparation of 3-O-deacylated monophosphoryl lipid A (3D- MLA/AF) according to the subject invention comprising 1000 ⁇ g ml 3D-MLA
• DPPC Dipalmitoyl- sn-glycero-3-phosphocholine
• Example 2-Lyophilization and Reconstitution of the Aqueous Formulation Two percent glycerol was added to an aqueous formulation of MLA prepared as in Example 1 The mixture was aliquoted in vials at volumes from one to 10 ml Nials were frozen in a lyophilizer at a shelf temperature of -45 °C.
• Table 1 shows that the aqueous formulation of the subject invention is restored to its original particle size after lyophilization by the addition of water.
• Example 3 Stimulation of an Antibody Response.
• mice immunized with tetanus toxoid (TT) in the aqueous formulation of the subject invention generated tetanus toxoid specific antibody.
• the TT-specific total IgG titer and IgG isotypes (2a, 2b, 1) titers were measured by enzyme-linked immunosorbent assay (ELISA) in the sera of mice following immunization.
• mice Female ICR mice were immunized with a dose of vaccine containing 0.1 ⁇ g of tetanus toxoid (TT) + 50 ⁇ g 3D-MLA/AF or 0.1 ⁇ g TT in saline.
• 3D-MLA/AF was prepared as in Example 1. The vaccines were administered by subcutaneous injection on days 0 and 21. Serum was collected 14 days post secondary immunization and assayed by standard ELISA techniques to report the relative amounts of tetanus-toxoid specific antibody of IgG 1; IgG 2a and IgG 2b isotypes as well as total IgG.
• Figure 1 shows the tetanus toxoid specific antibody titer generated by 3D- MLA/AF.
• 3D-MLA AF when administered with the tetanus toxoid antigen stimulates the production of IgG antibody in immunized animals and in particular actively stimulates IgG 2a production.
• PPD purified protein derivative
• mice Female BALB/c mice were immunized by subcutaneous injection with a dose of vaccines containing 50 ⁇ g PPD + 50 ⁇ g 3D-MLA/AF.
• 3D-MLA AF was prepared as in Example 1.
• Spleen cells were harvested 14 days after immunization and used as a source of lymphocytes in a proliferation assay.
• the spleen cells were cultured for 96 hr in microtiter wells at a concentration of 10 6 cells/ml in media containing 0.1, 1 or 10 ⁇ g PPD/ml. Tritiated thymidine was added to the cultures during the final 24 hr of incubation.
• the cells were harvested on glass fiber filters and tritium incorporation was determined.
• Stimulation indices were determined by dividing counts per minute (CPM) of cells stimulated with PPD by the CPM of cells cultured in media alone. The resulting data are shown in Figure 2.
• CPM counts per minute
• OVA OVA formulated in 3D-MLA/AF.
• 3D-MLA/AF was prepared as in Example 1. The injected volume was 200 ⁇ l. Twenty-one days later three mice per experimental group were killed and spleens removed and pooled as single cell suspensions and counted. Spleen cells (75 X 10 6 cells in 3-4 ml media) from the experimental groups were placed in a 25 cm 2 T-flask. Next, 1.0 ml of irradiated (20,000 rads) E.G7 (OVA) cells at 5 X 10 6 /ml were added to the flask. The volume was brought to 10 ml.
• the cultures were maintained by placing the T-flasks upright in a 37°C, 5% CO 2 incubator for four days. On day 4 the surviving cells were recovered from the flasks, washed IX, resuspended in 5.0 ml, and counted.
• Recovered effector cells were adjusted to 5 X 10 6 viable cells/ml and 100 ⁇ l volumes were diluted serially in triplicate in wells of 96 well round-bottom plates (Corning 25850) using 100 ⁇ l/well of media as a diluent.
• 100 ⁇ l volumes of 3l Cr-labelled (see below) targets [E.G7 (ONA)-an ovalbumin gene transfected EL-4 cell line] at 1 X 10 5 cells/ml were added to the wells.
• Spontaneous release (SR) wells contained 100 ⁇ l of targets and 100 ⁇ l of media.
• Maximal release (MR) wells contained 100 ⁇ l of targets and 100 ⁇ l detergent (2% Tween 20).
• Effector/target (E/T) ratios were 50: 1, 25: 1, 12.5:1, 6.25: 1.
• the plates were centrifuged at 400 Xg and incubated at 37° C, 5% CO 2 for 4 hr. After the incubation the well supernatants were collected using a Skatron Supernatant Collection System.
• Target cells E.G7 (ON A)
• 51 Cr sodium chromate
• Example 6-Stimulation of an Antibody Response by Intranasal Administration of the Aqueous ALD formulation Example 6-Stimulation of an Antibody Response by Intranasal Administration of the Aqueous ALD formulation.
• PBS phosphate buffered saline
• mice were primed on day 0, bled on day 10 (dlOPl °), boosted on day 14, bled on day 24 (dl0P2°), boosted on day 28, bled on day 38 (dl0P3 °).
• ELISA for IgG- and IgA specific anti-tetanus toxoid antibody was done on pooled sera from each bleed. Fecal extracts were examined on day 22 (d7P2°). IgG and IgA titers of sera and fecal extracts of immunized mice are shown in Tables 3-6. Table 3
• mice administered hepatitis B surface antigen (HBSAG) in the composition of the subject invention intranasally produced serum IgG and IgA titers to that antigen.
• Secretory IgA was detected in vaginal washes and the induction of a cytotoxic T-lymphocyte response was detected by a cytotoxicity assay.
• mice Groups of Balb/C mice were given a primary immunization (1 °) intranasally with 2.5 ⁇ g HBsAg + 10 ⁇ g 3D-MLA/AF in a volume of 20 ⁇ l. 3D-MLA/AF was prepared as in Example 1. Twenty-one days later mice were given a secondary immunization (2°) of 7.5 ⁇ g HBsAg + 10 ⁇ g 3D-MLA/AF intranasally in 20 ⁇ l. A tertiary immunization (3°) identical in composition to the secondary immunization was administered 28 days after the secondary immunization.
• mice were immunized with 2.5 ⁇ g HBsAg + 10 ⁇ g 3D- MLA/AF intranasally and boosted intranasally with 7.5 ⁇ g HBsAg + 10 ⁇ g 3D- MLA/AF 21 days later. Vaginal samples were collected 10 days after the booster immunization. Table 9
• HBsAg intranasal administration of HBsAg in the composition of the subject invention stimulated both a humoral and cellular immune response to that antigen.
• Intranasal immunization with the antigen formulated in 3D-MLA/AF induced a cytotoxic T-lymphocyte response and antigen specific humoral and mucosal immune responses.
• mice immunized intranasally with FLUSFflELD influenza vaccine containing hemagglutinin antigen formulated in the composition of the subject invention produced both IgG and IgA which were recovered in vaginal washes. Immunized mice were also protected 100% from subsequent influenza challenge.
• ICR mice were immunized three times at 21 day intervals intranasally with FLUSHIELD influenza vaccine (Wyeth-Lederle) containing 0.3 ⁇ g hemagglutinin antigen (HA) + 10 ⁇ g 3D-MLA/AF. 3D-MLA/AF was prepared as in Example 1. Vaginal washes were collected 14 days after the final immunization. Mice were challenged with 10 LD 50 (lethal dose 50) of infectious influenza A/HK/68 thirty-five days after the final immunization and monitored for mortality. Table 10
• Example 9-Compositions of Monophosphoryl Lipid A Monophosphoryl lipid A (MLA) can be formulated into the aqueous compositions of the subject invention and administered in the same quantities and amounts as in Examples 1-7 to produce similar results.
• MVA monophosphoryl lipid A

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• 1999
  • 1999-06-22 US US09/338,182 patent/US6491919B2/en not_active Expired - Lifetime
• 2000
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