Patent Application
20070184047
The present invention relates to methodology and compounds useful to augment the efficacy and in instances replace IVIg and anti-D therapies for immune cytopenias. More specifically, the present invention relates to the use of nitrophenyl compounds and thimerosal and congeners thereof in methodology for immune cytopenia treatments and treatment of autoimmune tissue diseases.
It is well established in the field that IVIg and anti-D are derived from human source material. This obviously presents health risk and economic issues. The danger for transmission of infectious blood diseases clearly exists, which is exacerbated by significant side effects attributable to use. In respect of the economics, extraction and other processing of the compounds is involved and given that large quantities (grams per kilogram of body weight) are necessary for treatment, costs escalate commensurately.
Rampersad et al, in Transfusion, Volume 45, March 2005, investigated the in vitro affects of nitrophenyl compounds as related to specific sulfur redox reactions. The conclusion was drawn that mechanisms which target sulfhydryl groups on mononuclear phagocytes may present therapeutic benefit in the treatment of immune cytopenias.
It has been recently shown that certain chemical compounds containing para-nitrophenyl and sulfur-reactive substituent groups can inhibit FcγR-mediated phagocytosis in vitro and may pose promising drug candidates for future treatment of immune cytopenias, Rampersad et al., in Transfusion, 2005; 43:1-9; and Foo et al. in Transfusion, 2007; in press. The mechanism of action of these compounds has been proposed to involve indirect interference of the interaction of FcγR with antibody-coated red blood cells by steric hindrance after binding to thiol groups on the surface of monocyte-macrophages (Mφ) within close proximity to FcγRs, Woodruff et al., Lancet, 1986; 2:217-8. Immunoglobulins, in contrast, have been shown to inhibit FcγR interaction with antibody-coated cells by directly binding to the FcγR resulting in ‘blockade’ of this interaction.
What is currently absent from the immunohematology field is a group of compounds and protocol for the use of these for the treatment of autoimmune or alloimmune diseases. The present invention is directed to addressing this need.
Accordingly, an object of the present invention is to provide a method for inhibiting phagocytosis of blood cells, comprising providing a nitrophenyl compound and administering said compound to a host having an auto or alloimmune disease for the inhibition of said phagocytosis of blood cells such as red cells, platelets and granulocytes. Examples of the auto or alloimmune disease include, but are not limited to thrombocytopenia, hemolytic anemia, immune cytopenia, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, inter alia.
Another object of one embodiment of the present invention is to provide a method for inhibiting phagocytosis of blood cells, comprising providing a thimerosal compound and administering said compound to a host having an auto or alloimmune disease for the inhibition of said phagocytosis of blood cells.
A further object of one embodiment of the present invention is to provide a composition for inhibiting phagocytosis of blood cells, comprising p-nitrophenyl methyl disulfide.
According to another object of one embodiment of the present invention there is provided a composition for inhibiting phagocytosis of blood cells, comprising p-nitrophenylethanol.
According to a still further object of one embodiment of the present invention there is provided a composition for inhibiting phagocytosis of blood cells, comprising thimerosal.
A still further aspect of one embodiment of the present invention is to provide a composition for inhibiting phagocytosis of blood cells, comprising an immunoglobulin preparation selected from at least one of IVIg or anti-D and a nitrophenyl compound or thimerosal, said preparation and said compound being present in an amount sufficient to effect inhibition of said phagocytosis.
A further object of one embodiment of the present invention is to provide a method for inhibiting tissue destruction due to an autoimmune disease, comprising providing a nitrophenyl compound or thimerosal to a host having an autoimmune disease for the inhibition of said tissue destruction.
These and other features, aspects and advantages of the present invention will become better understood with regard to the following description and accompanying drawings.
It will be noted that throughout the appended drawings, like features are identified by like reference numerals.
The human monocytic cell line THP-1 (ATCC 202, Manassas, Va., USA) was maintained in continuous culture in RPMI-1640 (Gibco/Invitrogen, Burlington, Ontario, Canada) containing 10% FBS (Sigma-Aldrich, Oakville, Ontario, Canada) and 0.1% gentamycin (Gibco/Invitrogen) at 37° C. and 5% CO2. THP-1 is a non-adherent leukemia cell line that is phagocytic and contains FcγRs but no cytoplasmic immunoglobulins, Tsuchiya et al., Int. J Cancer, 1980; 26(2):171-6. Normal human peripheral blood was obtained from volunteers. Thimerosal was purchased from BioShop Canada Inc., Burlington, ON, Canada. Human polyclonal anti-D for Slide and Tube Reagent Tests was obtained from Immucor, Houston, Tex., USA. WinRho SDF anti-D was obtained from Cangene. GAMMAGARD S/D Immunoglobulin Intravenous (Human) Therapy (IVIg) (Baxter, Ill., USA) was obtained from Canadian Blood Services.
The test concentration of immunoglobulin (anti-D or IVIg) was chosen after titration of each immunoglobulin on its own to inhibit FcγR and phagocytosis of anti-D-coated RBCs using a monocyte monolayer assay (MMA) as previously described in Rampersad et al., supra and Foo et al., supra. Based on these dose-response inhibitory titration curves, a concentration for each immunoglobulin was chosen so as to have a half-maximum (50%) inhibitory effect. These concentrations were a ⅙ dilution for slide and tube reagent test anti-D, 0.025×10−3 mg/ml for WinRho SDF anti-D, and 0.05 mg/ml for IVIg (
Preparation of anti-D-sensitized R2R2 red blood cells (RBCs) has been previously described in Rampersad et al., supra and Foo et al., supra. Briefly, RBCs were resuspended in PBS to 5% concentration, mixed with an equal volume of human polyclonal anti-D (Immucor, Tex., Houston, USA) in PBS solution, and then incubated for 1 hour at 37° C. and 5% CO2. After which, the sensitized RBCs were washed four times in PBS and resuspended to 2.5% in PBS. Before the RBC suspension was mixed with an equal volume of culture medium (RPMI-1640 (Gibco/Invitrogen) supplemented with 10% (vol/vol) fetal bovine serum (Sigma-Aldrich) and 20 mM Hepes buffer, pH 7.4 (Gibco/Invitrogen)), an indirect antiglobulin test (IAT) was performed to assess the level of antibody coating of the RBCs and yielded a 4+ reaction. The MMA was performed as previously described in Foo et al., supra, without modification.
A phagocytic index was calculated as described in Rampersad et al., supra; Foo et al., supra; and Branch et al., British Journal of Haematology, 1984; 56:19-29, as the unitless number of antibody-sensitized RBCs phagocytosed per 100 Mφ. Residual and phagocytosed RBCs were distinguished by relative differences in refracted light under phase-contrast microscopy. Percent inhibition was calculated as previously described2 taking the phagocytic control index to be 100. The means and standard error of the mean (SEM) of the results from several independent experiments were determined and analysed statistically. Statistical significance of inhibition between treated and untreated Mφ were analysed using Student t-test and Analysis of Variance (ANOVA), and/or, General Linear Model (GLM) Analysis and Student-Newman-Keuls test. A p value of <0.05 was considered to be significant.
Thimerosal at 10−5M or 10−3M was mixed with slide and tube reagent anti-D used at a ⅙ dilution as illustrated in
As shown in
Chemically treating different preparations of anti-D with thimerosal was found to enhance the ability of anti-D to inhibit in vitro phagocytosis by up to 100% (
Structure—Functional Analysis—In Vitro Chemical Inhibition of FCγ-Receptor-Mediated Phagocytosis
Preparation of p-nitrobenzyl methyl sulfide
Sodium metal (0.53 g, 23 mmol) was dissolved in methanol (100 mL) and p-nitrobenzyl mercaptan (3.94 g, 23 mmol) was added. The deep red solution was cooled with an ice-water bath. Methyl iodide (3.98 g, 28 mmol) in methanol (10 mL) was added dropwise over 3 minutes. The purple reaction mixture was stirred at ambient temperature for 24 hours.
Water (110 mL) was added and the resultant mixture was extracted with chloroform (three 100-mL aliquots). The combined organic layers were dried (MgSO4) and filtered and the solvent was evaporated. Crude product was chromatographed on silica gel (400 g) employing 2:1 petroleum ether:chloroform (100 mL fractions). Fractions 36 to 57 were combined and concentrated, affording impure p-nitrobenzyl methyl sulfide (1.45 g). Impure chromatographed product was rechromatographed on a four-bundle system, Kabir et al., J. Sulfur Chem, 2005; 26:7-11, employing petroleum ether. Fractions 155 to 209 were combined and concentrated, affording clean p-nitrobenzyl methyl sulfide (0.46 g, 2.5 mmol, 11%). p-Nitrobenzyl methyl sulfide had infra-red 1519 and 1347 per cm. 1H nuclear magnetic resonance (270 MHz): δ 1.98 (s, 3H), 3.72 (s, 2H), 7.45 (d, 2H), 8.17 (d, 2H). Gas chromatography-mass spectrometry (Rt=8.5 min): 183 (100%, M+), 136 (98%), 106 (38%), 89 (53%), 78 (60%).
Preparation of benzoylmethyl mercaptan
Benzoylmethyl methyl disulfide or phenacyl methyl disulfide, Griffiths et al., Aust J Chem, 2005; 53:1-5, (0.21 g, 1.06 mmol) was added to dry methylene chloride (4 mL). Thiophenol (0.25 g, 2.27 mmol) and pyridine (0.1 mL) were added to the reaction mixture. The resultant solution was stirred at ambient temperature for 2 hours. The solvent was evaporated and the mixture was chromatographed on silica gel (10 g) employing 3:2 petroleum ether:chloroform (5 mL fractions) for elution. Fractions 7 to 15 were combined and concentrated.
The concentrate was dissolved in chloroform (50 mL) and the resultant solution was extracted with 2.5 percent (w/v) sodium hydroxide (four 25-mL aliquots). The combined aqueous layers were set aside, the organic layer was dried (MgSO4) and filtered, and the solvent was evaporated, affording unchanged benzoylmethyl methyl disulfide (0.077 g, 37%).
The combined aqueous layers were acidified with concentrated hydrochloric acid (15 mL) and the resultant was mixture extracted with chloroform (four 50-mL aliquots). The combined organic layers were dried (MgSO4) and filtered and the solvent was evaporated yielding benzoylmethyl mercaptan (0.091 g, 0.59 mmol, 56%). Benzoylmethyl mercaptan had infrared 2580, 1680 per cm. 1H nuclear magnetic resonance (270 MHz): δ 2.14 (t, 1H, J=8.1 Hz), 3.96 (d, 2H, J=8.1 Hz), 7.48 (t, 2H), 7.60 (t,1H), 7.96 (d, 2H). 13C nuclear magnetic resonance: δ 31.15, 128.51, 128.82, 133.63, 135.01, 194.74. Gas chromatography-mass spectrometry (Rt=6.25 min): 105 (100%), 77 (62%).
RBC Sensitization
As previously described with minor modification, Rampersad et al., Transfusion, 2005; 45:384:93, an aliquot of R2R2 RBCs was removed from storage in Alsever's solution, Walker et al., American Association of Blood Banks, 11th ed., Bethesda, 1993, at 4° C. and washed three times in PBS without calcium or magnesium (Gibco/Invitrogen) at 2000 r.p.m. for 7 minutes (Sorvall RT 6000D centrifuge, Mandel Scientific Company Inc., Guelph, Ontario, Canada). The RBCs were resuspended in PBS to 5 percent concentration, mixed with an equal volume of human anti-D in PBS solution, and then incubated for 1 hour at 37° C. and 5 percent CO2 (Sanyo CO2 incubator), after which the sensitized RBCs were washed four times in PBS at 2000 r.p.m. for 7 minutes and resuspended to 2.5 percent in PBS. Before the RBC suspension was mixed with an equal volume of culture medium, an indirect antiglobulin test was performed to assess the level of antibody coating of the RBCs and yielded a 4+ reaction.
Monocyte Monolayer Assay
As previously described with slight modification to improve the quality of the Mφ monolayer, Rampersad et al, supra, whole venous blood was drawn from donors into tubes (Vacutainer, ACD solution A, Becton Dickinson Vacutainer Systems, Franklin Lakes, N.J.) and mixed with an equal volume of PBS. In 50-mL tubes (Sarstedt Inc., Montreal, Quebec, Canada), 35 mL of the blood mixture was overlayed onto 15 mL of Ficoll-Paque separation medium (GE Healthcare, Baie d'Urfé, Quebec, Canada), and peripheral blood mononuclear cells (PBMCs) were isolated with density gradient centrifugation at 1800 r.p.m. for 25 min. The PBMCs were washed three times in PBS heated to 37° C., centrifuged at 1200 r.p.m. for 15 minutes and resuspended in culture medium. Viable cell concentration was then adjusted to approximately 2×106 cells per mL. One milliliter of this suspension was overlayed onto each 22×22-mm coverslip (Fisher Scientific, Waltham, Mass.) in respective 35 mm petri dishes (Sarstedt Inc.). After 1 hour of incubation at 37° C. and 5 percent CO2, coverslips were washed in PBS that had been warmed to 37° C. and placed in new petri dishes with the mononuclear monolayer side facing upward. One milliliter of chemical solution was then overlayed onto each coverslip. For each chemical, the concentrations 10−4, 10−5, 10−6, 10−7, 10−8, and 10−9 mot per L were tested in triplicate. For the positive control, culture medium alone was used in place of drug treatment. Following another incubation period of 1 hour, coverslips were washed gently in 37° C. PBS, transferred to new petri dishes with the monolayer side facing upward, and overlayed each with 1 mL of anti-D-sensitized RBC solution. The cells were then incubated for 2 hours at 37° C. and 5 percent CO2, washed gently in 37° C. PBS, and air-dried, after which coverslips were first fixed with methanol and then mounted to glass slides with elvanol (20 g of polyvinyl alcohol resin [Sigma-Aldrich] was dissolved in 80 mL PBS at 70° C. in a water bath. Afterward, the solution was cooled and mixed thoroughly with 40 mL of glycerin (ICN Biomedicals, Inc., Aurora, Ohio; final pH was between 6.6 and 7.0). Visual analysis was per-formed by phase contrast microscopy as described previously, Rampersad, Supra.
Fluorescence-Activated Cell Sorting Analysis for Viable and Apoptotic Cells
Fluorescence-activated cell sorting (FACS) analysis (flow cytometry) and a TACS annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (R&D Systems, Minneapolis, Minn.) were used to determine if disulfide-containing compounds benzoylmethyl methyl disulfide and phenyl methyl disulfide exert effects on viability or apoptosis of PBMCs. Briefly, PBMCs were isolated from whole blood as previously outlined and treated with culture medium or chemical from 10−4 to 10−9 mol per L for 1 hour at 37° C. and 5 percent CO2. They were then washed three times in PBS and collected by centrifugation at 12,000 r.p.m. for 5 to 10 minutes before being incubated in culture medium for 2 hours. After washing the cells once in cold PBS, according to the manufacturer's instructions, cells were then incubated with annexin V-FITC incubation reagent for 15 minutes at room temperature to stain membrane exposed phosphatidylserine, indicating early programmed cell death or apoptosis. Cells were then stained with propidium iodide (PI), specific for nonviable cells, to identify late apoptotic cells. FACS was performed with two-color analysis on a flow cytometer (FACSCalibur E4795, Becton Dickinson, Mississauga, Ontario, Canada) calibrated with fluorescent beads (CaliBRITE, BD Biosciences, San Jose, Calif.) and computer software for data analyses (Cell Quest, BD Biosciences).
To establish that a disulfide bond is one requirement for a compound to have efficacy for FcγR blockade, the activities of benzoylmethyl methyl disulfide and benzoylmethyl mercaptan were tested and compared as illustrated in
To further confirm the importance of a disulfide moiety, the lead compound selected was p-nitrophenyl methyl disulfide. Activity was compared to that of a structurally similar compound synthesized to lack the disulfide moiety, p-nitrobenzyl methyl sulfide (
To establish that the phenyl group itself induces efficacy for FcγR blockade that is further enhanced by the nitro group, we have tested phenyl methyl disulfide (
To examine the importance of the reactive p-nitrophenyl group and to further elucidate other potentially reactive groups as involved in the chemical inhibition of FcγR-mediated phagocytosis, compounds completely lacking sulfur but containing various combinations of the functional groups were tested, p-nitrophenyl and/or hydroxyl moieties (
By use of an annexin V-FITC apoptosis detection kit (R&D Systems) and FACS analysis, it was determined that disulfide compounds, benzoylmethyl methyl disulfide and phenyl methyl disulfide, do not significantly affect PBMC viability or apoptosis. Even at concentrations as high as 10−4 mol per L, benzoylmethyl methyl disulfide-treated PBMCs had viable cell counts comparable to control untreated PBMCs as indicated in the lower left quadrants (
It has been demonstrated herein that the presence of a disulfide bond is important. As disulfide groups react with free sulthydryl groups, it is believed that any compound that reacts with free sulfhydryl groups has the potential to inhibit FcγR-mediated phagocytosis. A p-nitrophenyl group provides enhancement to the efficacy of disulfide-containing compounds.
Mercaptan conjugate bases react with disulfides as shown in Scheme 1.
Referring now to
Referring to
It can be seen that the compound p-nitrophenylethanol (4-nitrophenyl-OH) administered prior to the anti-platelet antibody results in zero platelet loss. In fact, this compound works better than IVIg (2 g/kg). The figure is representative of three such experiments having three mice in each group.
It will be understood that numerous modifications thereto will appear to those skilled in the art. Accordingly, the above description and accompanying drawings should be taken as illustrative of the invention and not in a limiting sense. It will further be understood that it is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein before set forth, and as follows in the scope of the appended claims.
The embodiments of the invention described above are intended to be exemplary only. The scope of the invention is therefore intended to be limited solely by the scope of the appended claims.
This application claims benefit of U.S. Provisional Application No. 60/752,912, filed Dec. 23, 2005.
| Number | Date | Country | |
|---|---|---|---|
| 60752912 | Dec 2005 | US |
