Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/415—1,2-Diazoles
• • 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

Definitions

• This invention relates to therapeutic agents and more particularly to immunoregulatory agents.
• aldose reductase catalyzes the reduction of glucose to sorbitol and has been implicated in processes leading to certain complications of diabetes, including cataracts, retinopathy, nephropathy, and peripheral neuropathy. For this reason, inhibitors of the aldose reductase enzyme are of considerable medical interest. For current reviews, see: Dvornik, D., Aldose Reductase Inhibition, McGraw-Hill, New York, N.Y., 1987; and, Polyol pathway and its role in diabetic complications, N. Sakamoto, et al., eds., Elsevier Science Publishers, New York, N.Y., 1988.
• sorbinil [4(S)-2,3-dihydro-6-fluorospiro-(4H-l-benzopyran-4,4'-imidazolidine)- 2',5'-dione].
• Sorbinil and its analogs defined herein as spirohydantoin derivatives wherein essentially planar benzene and hydantoin rings are connected via a nonplanar pyran ring, are the subject of the following United States patents, all of which are herein incorporated by reference: 4,127,665; 4,130,714; 4,147,795; 4,147,797; 4,181,728; 4,181,729; 4,235,911; 4,286,098; 4,248,882; 4,348,526; and 4,431,828.
• aldose reductase inhibitors whose molecular structures do not conform to the above-stated definition of sorbinil analogs are also known: tolrestat, N-[[6-methoxy-5-(trifluoromethyl)thio-l-napthalenyl]thioxomethyl]-
• N-methylglyeine flavonoids having the basic structure of 3-hydroxyflavone; acetylsalicylic acid (aspirin); diphenylhydantoin (DilantinTM); phenobarbital; sulindac; indomethacin; ICI 105552; alrestatin (AY-22,284); and WF-3681, 3-(2,5-dihydro-4-hydroxy-5-oxo-3-phenyl-2-furyl)propionic acid.
• Interactive atoms are defined as those that may participate directly in chemical mechanisms that include charge-transfer, hydrogen bonding, electrophilic and/or nucleophilic substitutions, and hydrophobic interactions. Interacting atoms include but are not limited to oxygen, sulfur, and nitrogen.
• aldose reductase inhibitors were known to also act as anti-inflammatory (Al) agents: flavonoids having the structural backbone as in
• certain aspects of the cellular and humoral immune systems of a mammalian host are specifically regulated by administering to the host a compound having a reactive immunoregulatory conformation whose precise three-dimensional structure is defined below.
• Sorbinil, tolrestat, WF-3681, and the other immunoregulatory agents described herein possess this reactive conformation and so are therapeutically useful for inhibiting delayed-type hypersensitivity, T-cell proliferation, and B-cell antibody production, without altering the normal cellularity of the immune system or the normal metabolites of the arachidonic acid pathway.
• FIGURE 1 is a diagrammatic frontal view of the immunoregulatory conformation, with plane P ⁇ , parallel to the plane of projection;
• FIGURE 2 is a diagrammatic edge view in which the immuno ⁇ regulatory conformation shown in FIGURE 1 has been rotated so that plane P j is perpendicular to the plane of projection, and so that reference atom 8 is projected toward the viewer;
• FIGURE 3 is a diagrammatic edge view in which the immuno ⁇ regulatory conformation shown in FIGURE 2 has been rotated so that both of planes P j and P 2 are perpendicular to the plane of projection, and so that both of reference atoms 8, 10 are projecting toward the viewer;
• FIGURE 4 depicts the immunoregulatory conformation of FIGURE 1 superimposed upon the molecular conformation of sorbinil;
• FIGURE 5 depicts the immunoregulatory conformation of FIGURE 2 superimposed upon the molecular conformation of sorbinil;
• FIGURE 6 depicts the immunoregulatory conformation of FIGURE 1 superimposed upon the molecular conformation of the deaeetylated form of aspirin;
• FIGURE 7 depicts the immunoregulatory conformation of FIGURE 2 superimposed upon the molecular conformation of the deaeetylated form of aspirin
• FIGURE 8 depicts the immunoregulatory conformation of FIGURE 1 superimposed upon the molecular conformation of the acetylated form of aspirin;
• FIGURE 9 depicts the immunoregulatory conformation of FIGURE 2 superimposed upon the molecular conformation of the acetylated form of aspirin;
• FIGURE 10 depicts the immunoregulatory conformation of FIGURE 1 superimposed upon the molecular conformation of 3-hydroxyflavone
• FIGURE 11 depicts the immunoregulatory conformation of FIGURE 2 superimposed upon the molecular conformation of 3-hydroxyflavone
• FIGURE 12 depicts the precise three-dimensional conformation of sorbinil, as described in Experiment 1 herein;
• FIGURE 13 is a graph which summarizes the results of Experiment 2 as described herein;
• FIGURE 14 is a graph which summarizes the results of Experiment 3 and shows that sorbinil inhibits the inflammatory response associated with the delayed-type hypersensitivity reaction
• FIGURE 15 is a graph which shows that sorbinil causes a dose dependent inhibition of the delayed-type hypersensitivity response;
• FIGURE 16 is a photomicrograph (approximately 700X) of a cross section of the pinna of a normal primed mouse that was not challenged with DNFB, as described in Experiment 5 herein;
• FIGURE 17 is a photomicrograph as in FIGURE 16 but of a buffer- treated control mouse that was challenged with DNFB;
• FIGURE 18 is a photomicrograph as in FIGURE 16 but of a sorbinil- treated primed mouse that was challenged with DNFB;
• FIGURE 19 is a photomicrograph as in FIGURE 16 but of a primed mouse treated with sorbinil but not challenged with DNFB;
• FIGURE 20 is a graph which shows that sorbinil inhibits the proliferative response of T-cells to the mitogen Con A, as described in Experiment 8 herein;
• FIGURE 21 is another graph which shows that sorbinil inhibits the proliferative response of T-cells to the mitogen Con A, as described in Experiment 8 herein.
• the invention is predicated upon applicants' discovery that the mammalian immune system can be therapeutically perturbed in a previously undisclosed manner, by a family of compounds having a reactive conformation whose precise three-dimensional structure is defined below.
• These immunoregulatory agents do not affect the normal cellularity of the immune system in an unstimulated animal, but only regulate an immune system that is stimulated by pathological conditions.
• Such regulation includes inhibition of the delayed-type hypersensitivity (DTH) reaction, inhibition of T-cell mitogen responsiveness, and inhibition of antibody production in response to antigenic challenge.
• DTH delayed-type hypersensitivity
• T-cell mitogen responsiveness inhibition of antibody production in response to antigenic challenge.
• these effects are achieved without altering, in unstimulated animals, the normal metabolites of the arachidonic acid pathway. It is particularly noteworthy that the described regulatory effects are achieved when the subject compounds are administered either before or after the host's immune system is pathologically stimulated.
• the subject compounds can be used to regulate specific aspects of the immune system, by inhibiting delayed-type hypersensitivity, T-cell proliferation, and B-cell antibody production.
• these compounds are of therapeutic value in the treatment of autoimmune diseases, including periodontal disease and rheumatoid arthritis, allergic reactions, inflammatory dermatitis, hypersensitivity reactions, inflammation, undesirable side effects of immunization, graft rejection, and graft vs. host complications occurring in bone marrow transplantation.
• an aromatic ring 12 is associated in defined conformation with two distinct interactive atoms X j , X .
• the aromatic ring 12 is essentially planar, being composed of five to seven atoms (a representative six-membered ring is shown) that are cyl ⁇ ndrically symmetrical within about 15 degrees of planar and preferably within about seven degrees or less of planar.
• the aromatic ring 12 can be homocyclic or heterocyclic.
• Representative rings 12 include benzene, pyridine, furan, and thiophene.
• Aromatic ring 12 may be part of a ring complex, e.g., a naphthalene or anthracene derivative.
• the interactive atoms Xp X 2 can each be an oxygen, sulphur, or nitrogen atom.
• Each interactive atom Xp X 2 may be a substituent of a hydantoin ring, ether, or other group that disposes the interactive atom in the defined conformation.
• the aromatic ring 12 (or ring complex) may have fluorine, chlorine,
• P 2 the plane defined by the two interactive atoms Xp X 2 and the center 14 of the aromatic ring 12,
• the reactive conformation of the immunoregulatory compound is defined with respect to the aforementioned parameters as follows:
• FIGURE 2 another view of the above-defined reactive conformation is shown, here in edge view in which FIGURE 1 has been rotated so
• P j and P 2 is illustrated, here in edge view in which FIGURE 2 has been rotated so that both of planes P, and P are perpendicular to the plane of projection, and so that both of reference atoms 8, 10 of the aromatic ring 12 are projecting toward the viewer.
• the immunoregulatory compound has a reactive conformation that is defined with reference to FIGURES 1, 2, and 3 and the aforementioned parameters as follows: D j equals approximately 4.06 A, D 2 equals approximately 2.74 A,
• the immunoregulatory compound has a reactive conformation that is defined as follows:
• the subject immunoregulatory compound has a reactive conformation that is defined as follows:
• D j equals approximately 3.97 A
• D 2 equals approximately 2.45 A
• D equals approximately 4.10 A
• ⁇ ° equals approximately 47.0°.
• TABLE 2 summarizes the relationship between the precise three- dimensional molecular structures of sorbinil, tolrestat, and WF-3681, and their unique i munoregulatory properties.
• aldose reductase inhibitors ARI
• their other pharmaceutical activities anti-inflammatory (Al), anti- convulsant (AC), and immunoregulatory (IR) as defined herein; the underlined activities are pursuant to this disclosure.
• the coordinates and parameters of the immunoregulatory conformation are presented in the five right columns.
• Dp the distance between the center 14 of P-, and interactive atom Xp because sorbinil has two oxygens (O ⁇ and O j g in FIGURES 4, 5, and 12) that could potentially participate in aldose reductase inhibitory activity.
• Aspirin also has two carbonyl oxygens (O j and O 4 in FIGURES 6, 7, 8, and 9) that could participate in aldose reductase inhibitory as well as anti-inflammatory activity. It is well known that aspirin is deaeetylated to salicylate, and thereby loses one of the carbonyl oxygens (O ⁇ ), when administered clinically. TABLE 2 thus includes structural parameters for both the acetylated and deaeetylated forms of aspirin. The flavone structure listed is 3-hydroxyflavone. Several flavonoid analogs are known to exhibit anti- inflammatory activity, and all of them have the same basic three-dimensional conformation as 3-hydroxyflavone.
• aldose reductase inhibitory activity depends upon the presence of an aromatic ring 12 and an interactive atom X ⁇ in a carbonyl group, as was hypothesized in the prior art;
• anti-convulsant activity of several aldose reductase inhibitors is associated with the presence of a hydantoin ring that contains the interactive atom X- ⁇ that is associated with the aromatic ring 12;
• anti-inflammatory and immunoregulatory activities of certain aldose reductase inhibitors are specifically determined by a conformational relationship between an aromatic ring 12, an interactive atom X j in a carbonyl group, and a second interactive atom X 2 , all three of which define a plane P 2 with a specific spatial and angular orientation to the plane P ⁇ of the aromatic ring 12; and 4. the immunoregulatory compounds are distinguished from the anti-inflammatory compounds by having the defined reactive conforma A ",.
• interactive atoms X-, and X 2 are separated by distances D3 that are also tabulated in TABLE 2.
• the interactive atoms X j and X 2 form a second plane, P 2 , with the center 14 of plane Pp
• the angular relationship between planes P-- and P 2 is described by the angle, ⁇ °, between vectors 16, 18 constructed normal to each of planes Pi and P 2 (see
• FIGURE 3 These values for alpha are also listed in TABLE 2.
• D 2.74 for either, while Dg equals 4.00 A or 6.41 A, and ⁇ ° equals 38.85° or 9.89°, respectively.
• sorbinil may exhibit anti-inflammatory in addition to aldose reductase inhibitory activity.
• dissimilarity in the ⁇ ° values suggested that the nature of anti-inflammatory activity of sorbinil might be different than that of the other agents.
• FIGURES 4 through 11 depict the molecular conformations for sorbinil and the aldose reductase inhibitors that have anti-inflammatory activity. Note that the model represented in FIGURES 1 and 2 is superimposed over the molecular structures of the compounds in FIGURES 4 through 11.
• FIGURES 4 and 5 two views of the sorbinil molecule demonstrate the immunoregulatory reactive site of the present invention.
• a front view of sorbinil shows the aromatic plane P, and the plane P 2 formed by the center 14 of P ⁇ and the carbonyl oxygen at X_ and the ether oxygen at X 2 .
• the distance coordinates D D 2 , and D are also indicated.
• FIGURE 5 is an edge view of sorbinil diagrammed so that X (O j ) is projected toward the viewer.
• FIGURE 5 clearly shows that P j and P 2 are not in the same plane.
• the carbonyl oxygen at X j is positioned above the plane P
• the larger values for D ⁇ and ⁇ ° exhibited by the O 15 (X- ) carbonyl are consistent with the three-dimensional conformation necessary for immunoregulatory activity.
• FIGURE 6 is a front view of deaeetylated aspirin.
• FIGURE 7 also shows the acetyl group of aspirin that is lost when deacetylation breaks the bond at position 20. Note that the acetylated aspirin has a carbonyl oxygen O 4 that occupies a potential X ⁇ position.
• FIGURES 8 and 9 show the acetylated form of aspirin.
• O 4 is the carbonyl at X ⁇ which forms plane P 2 with the center 14 of P j and the ether oxygen O3 at X 2 .
• ⁇ ° 86.87°.
• P 2 is nearly orthogonal to plane P j as shown in FIGURE 9.
• the acetylated form is not normally present in vivo due to the rapid hydrolysis of aspirin to salicylate in the blood.
• FIGURES 10 and 11 show the relationship between the immunoregu ⁇ latory model and the three-dimensional molecular structure of 3-hydroxyflavone. Planes P- ⁇ and P 2 of the flavone are very similar to planes P, and P 2 of the deaeetylated aspirin.
• ⁇ ° for flavone is 0.69, which is the same value as for deaeetylated aspirin and much less than the ⁇ ° of sorbinil. Again, these different ⁇ ° values suggest there might be potential differences between the anti- inflammatory activities of sorbinil and the flavones.
• Formulas 1 and 2 present the atomic numbering systems from which the coordinates of the reactive conformations for tolrestat and WF-3681 in TABLE 2 are taken.
• Formula 1 shows tolrestat, as disclosed in Varughese, K.I., et al., Can. J. Chem. 61:2137-2140, 1983, which is hereby incorporated by reference.
• aromatic ring 12 in the tolrestat molecule is represented by A, interactive atom X j by sulfur atom S2, and interactive atom X 2 by oxygen atom O2.
• Formula 2 shows the molecular structure of WF-3681, as disclosed in Kissinger, C.R., et al., Acta Cryst. C44:512-514, 1988, which is hereby incorporated by reference.
• aromatic ring 12 in the WF-3681 molecule is represented by C4-O3-C5-C6-C7, interactive atom X ⁇ by oxygen atom O2, and interactive atom X 2 by oxygen atom O4.
• DTH delayed-type hypersensitivity
• sorbinil also affects T-cell proliferation and B cell antibody production in unusual and surprising ways.
• at least sorbinil and WF-3681 do not alter the normal metabolites of the arachidonic acid pathway in the normal, unstimulated animal.
• Sorbinil, tolrestat, and WF-3681 are thus representative of a new class of immunoregulatory compounds that, like aspirin and flavones, have anti-inflammatory activity but that unlike aspirin and the flavones affect cellular and humoral components of the immune system.
• the sorbinil structure also includes a hydantoin ring, as in diphenylhydantoin and phenobarbital.
• hydantoin ring is important to anti- convulsant activity and, based upon this relationship, we predicted that sorbinil exhibits anti-convulsant activity in addition to immunoregulatory activity.
• the anti-convulsant activity of sorbinil has since been independently reported; see Hall, P.C., and K.L. Keim, Anticonvulsant activity of sorbinil and AL-1576: hydantoin-containing aldose reductase inhibitors, Federation Proceedings 46(3):433, 606A, 1987.
• sorbinil In sorbinil the pyran ring stabilizes the relationships between X j and X 2 , and between the planes P j and P 2 . We contemplate that this stability is important to the immunoregulatory activities of sorbinil.
• many of the analogs of sorbinil that have been reported to possess aldose reductase activity have molecular configurations that include the above-defined immunoregulatory conformation.
• the sorbinil analogs that possess immunoregulatory activity can be defined as the group consisting of those of the formulas:
• Y ⁇ is hydrogen and Y 2 is hydrogen, hydroxy, fluorine, chlorine, lower alkyl or lower alkoxy (each having from one to four carbon atoms); or Y j and Y 2 , when taken separately, are each chlorine, lower alkyl or lower alkoxy, and when taken together are -OCH 2 (CH 2 ) n O-; and n is zero or one; (USPN 4,147,795; USPN 4,130,714); — substituted benz- ⁇ , ⁇ -O of the formula:
• R j is chlorine, bromine, fluorine, or alkyl of one to three carbon atoms; one of R 2 and Rg is hydrogen; and the other of R and Rg is amino, monoalkylamino or dialkylamino, wherein each alkyl group has from one to three carbon atoms (USPN 4,248,882);
• W is hydrogen, methyl, methoxy, phenyl, phenoxy, fluorine, chlorine, or boron at the 6 or 8 position; and Ar is phenyl or phenoxy at the 6 or 8 position (USPN 4,181,729);
• Sorbinil and its foregoing listed analogs are useful as immunoregulators, and as such are of therapeutic value in the treatment of autoimmune diseases, allergic reactions, hypersensitivity reactions, inflammation, inflammatory dermatitis, undesirable side effects of immunization, graft vs. host rejection, and complications of bone marrow transplant.
• treatment is meant to include prevention and/or alleviation of sueh conditions.
• the subject immunoregulatory compounds may be administered to a human patient or other mammalian host in need of treatment by a variety of conventional routes of administration, including orally and parenterally. In general, these compounds will be administered at dosages between 1 and 250 rag per kg body weight of the subject to be treated per day. However, some variation in dosage will necessarily occur depending on the condition of the patient being treated, and the physician will, in any event, determine the appropriate dose for the individual patient.
• salts can be readily prepared from sorbinil and sorbinil analogs by conventional methods.
• such salts may be prepared by treating the sorbinil or sorbinil analog with an aqueous solution of the desired pharmaceutically acceptable metallic hydroxide or other metallic base and evaporating the resulting solution to dryness, preferably under reduced pressure.
• a lower alkanoic solution of the sorbinil or sorbinil analog may be mixed with an alkoxide of the desired metal, and the solution subsequently evaporated to dryness.
• the pharmaceutically acceptable hydroxides, bases, and alkoxides include those with cations that form metal salts with the acidic compounds of sorbinil and its analogs and that are nontoxic at the dosages administered to a patient in need of treatment.
• Suitable cations for this purpose include, but are not limited to, potassium, sodium, ammonium, calcium, and magnesium.
• the compounds may be administered alone or in combination with pharmaceutically acceptable carriers, in either single or multiple doses.
• suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions and various nontoxic organic solvents.
• the pharmaceutical compositions formed by combining the sorbinil or sorbinil analog with the pharmaceutically acceptable carrier are then readily administered in a variety of dosage forms, such as tablets, powders, lozenges, syrups, injectable solutions and the like.
• These pharmaceutical compositions can, if desired, contain additional ingredients such as flavorings, binders, excipients, and the like.
• tablets containing various excipients such as sodium citrate, calcium carbonate, and calcium phosphate may be employed along with various disintegrants such as starch, and preferably potato or tapioca starch, alginic acid, and certain complex silicates, together with binding agents such as polyvinylpyrrolidine, sucrose, gelatin, and acacia.
• disintegrants such as starch, and preferably potato or tapioca starch, alginic acid, and certain complex silicates
• binding agents such as polyvinylpyrrolidine, sucrose, gelatin, and acacia.
• lubricating agents such as magnesium stearate, sodium luaryl sulfate, and talc are often useful for tableting purposes.
• Solid compositions of a similar type may also be employed as fillers in salt and hard-filled gelatin capsules; preferred materials for this purpose include lactose or milk sugar and high molecular weight polyethylene glycols.
• the essential active ingredient therein may be combined with various sweetening or flavoring agents, colored matter or dyes, and, if desired, emulsifying or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin, and combinations thereof.
• diluents such as water, ethanol, propylene glycol, glycerin, and combinations thereof.
• solutions of the sorbinil or sorbinil analog in sesame or peanut oil or in aqueous propylene glycol may be employed, as well as sterile aqueous solutions of the corresponding water soluble pharmaceutically acceptable metal salts previously described.
• Such an aqueous solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
• aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes.
• the sterile aqueous media employed are all readily obtainable by standard techniques well known to those skilled in the art.
• the subject compounds, when formulated as described above, will typically be packaged with printed instructions specifying their use for regulating the immune system, e.g., by inhibiting the delayed type hypersensitivity reaction. In order to assist in a better understanding of the present invention, the results of various experiments are set forth below.
• DTH delayed type hypersensitivity
• mice Eight-week old female C57BL/6J mice (Jackson Labs) were divided into primed and unprimed groups.
• the five mice in the primed group were primed to 2,4-dinitrofluorobenzene (DNFB; Sigma Chemical Co., Grade 1, approx. 98%, Lot 129C-0065, No. d-6879) by painting their shaved ventral skins with 0.1 ml of 0.5% DNFB in ethano acetone (3:1).
• the five mice in the unprimed group were shaved and topically painted on their ventral skins with the ethanol:acetone (3:1) vehicle alone.
• the mice in both groups were challenged with DNFB four days later. A suitable patch of skin, the ear, was chosen for challenging.
• One ear was designated for challenging and the other to serve as a control.
• the thickness of each ear across the pinna was measured using micrometer calipers. The first measurements were taken four days after priming and are plotted as day 0 in FIGURE 13.
• each mouse was subjected to the following challenge regimen: one ear was challenged with one drop of 0.35% DNFB in olive oil, and the other (unchallenged control) ear was exposed to olive oil alone.
• the ear thicknesses (ET) of the challenged ear and the unchallenged ear were measured daily, and the swelling responses are plotted in FIGURE 13 as:
• mice would develop an inflammatory response to this novel antigen, and the primed and unprimed curves in FIGURE 13 served as controls with which any anti-inflammatory effect of sorbinil could be compared.
• C57BL/6J mice were primed to DNFB as previously described.
• concentrations 10 1.0, or 0.1 mg/kg body weight per mouse.
• the first sets of ear measurements were taken, and one ear of each mouse was challenged. Sorbinil was subsequently administered at the stated concentrations six hours before each daily measurement of ear thicknesses.
• FIGURE 14 the primed and unprimed curves from FIGURE 13 are reproduced as dotted lines, and the inflammatory responses of the three sorbinil-treated groups are plotted as solid lines. Swelling was inhibited in animals that received sorbinil. Moreover, there was a dose dependent inhibition of ear swelling: the sorbinil was more effective at inhibiting ear swelling at higher concentrations. Sorbinil at a concentration of 10 mg/kg resulted in almost complete inhibition of ear swelling, as evidenced by the general confluence of the
• the dose effect of sorbinil is characterized by presenting the data accumulated from three replicates of Experiment 3. DTH response (%) is plotted versus sorbinil concentration (mg/kg) on a semilogarithmic scale, where
• the data in FIGURE 15 is derived from three experiments using C57BL/6J mice. The measurements were taken at the time of maximal swelling, i.e., 2 hours after challenge, and each point represents at least 9 mice. The data show that inhibition of the inflammatory response is dose dependent and may vary linearly with the log of the sorbinil dosage. Extrapolations from a least squares fit line indicates that maximum inhibition of DTH response (no swelling) would occur at a concentration of 13.5 mg sorbinil/kg body weight. Sorbinil significantly inhibits the DTH response, and thus it may have potential as a new anti-inflammatory agent for therapeutic uses that include the control of contact dermatitis and allograft rejection.
• Experiment 4 Experiments 2 and 3 were repeated with Balb C NCI mice (Life Sciences, Inc., St. Moscowh, FL). A similar dosage dependent mediation of the classic DTH response was observed in the mice that received sorbinil.
• Experiment 5 Experiment 5
• FIGURES 16, 17, 18, and 19 the histologic features of the DTH reaction in ears from mice treated with buffer (FIGURES 16 and 17) or sorbinil (FIGURES 18 and 19) are shown.
• FIGURE 16 depicts the ear of a primed mouse which has not been challenged with DNFB.
• the epidermis 22 is devoid of mononuclear infiltrate 28, as is the dermal compartment 24.
• a layer of cartilage 26 is located in the center of the ear.
• FIGURE 17 represents the ear of a buffer-treated primed mouse which had been challenged 24 hours previously with DNFB. Note the pronounced edema in the dermal compartment of the skin 24 as well as the extensive mononuclear cell infiltrate (indicated by arrows 28) in both the dermis and epidermis.
• the histologic appearance of the sorbinil-treated primed mouse challenged 24 hours previously with DNFB is shown in FIGURE 18.
• the dermal compartment 24 is much less edematous and contained fewer mononuclear cells 28. There were also fewer mononuclear cells 28 in the epidermis 22 as well.
• FIGURE 19 the ear of a primed mouse treated with sorbinil but not challenged with DNFB is shown.
• the unchallenged ear is devoid of edema and does not contain infiltrating mononuclear cells.
• FIGURE 18 The histologic section in FIGURE 18 indicates that the inhibition of swelling by sorbinil is accompanied by a mild cellular infiltration. This mild response suggested that sorbinil may affect not only the secretion of inflammatory mediators, but also the recruitment of cells to the site of inflammation. To our knowledge this type of immunoregulation has never been observed.
• the macrophages appear to function in two capacities: (1) as accessory cells that present the novel antigen to and thereby specifically sensitize the T-lymphocytes (antigen recognition), and then (2) as effector cells in DTH reactions after responding to mediators produced by the sensitized T-lymphocytes. These two functions may be correlated with the heterogeneity of the macrophage population: Some macrophages (or macrophage progenitors) are excellent antigen presenting cells, while other populations of macrophages are more active as effector cells.
• T-cells can be sensitized to the antigen and activated. Activated T-cells proliferate and express IL-2 receptors, and some activated T-cells secrete IL-2. Activated T-cells also secrete other mediators, e.g., macrophage inhibition factor (MIF), interferons, and other soluble factors. Some of these T-cell products, e.g., MIF and ⁇ , ⁇ interferons, can activate macrophages.
• MIF macrophage inhibition factor
• the activated macrophages in turn secrete IL-1 (endogenous pyrogen), catabolic enzymes (e.g., collagenase, hylaronidase, neutral proteinase), and prostaglandins of the E-series.
• IL-1 endogenous pyrogen
• catabolic enzymes e.g., collagenase, hylaronidase, neutral proteinase
• prostaglandins of the E-series.
• macrophages are not only necessary for antigen presentation to T-cells but are also the effeetor cells that cause the symptoms of immunologically mediated inflammation by secreting IL-1, prostaglandins, and catabolic enzymes at the challenged site.
• the immune system involves a complex series of cellular interactions which regulate the production of lymphocytes and mononuclear phagocytes, as well as affecting their differentiated function.
• the interdependence of these different cell populations in the immune system is reflected by their obligatory interactions for effective function.
• Most T-eell function is dependent on the participation of macrophages for antigen presentation.
• B cells require helper T-cells in order to produce antibodies to most antigens; the helper T-cells in turn require the participation of macrophages or accessory cells for antigen presentation. Interactions between T lymphocytes and macrophages thus play a
• Sorbinil-treated mice exhibited a 94% increase in the number of direct plaques, which are indicative of the number of IgM antibody secreting cells, when compared to the buffer control group: 372 ⁇ 62 plaques per 2 x 10 cells in the sorbinil group; 191 ⁇ 50 plaques per 2 x 10 cells in the control group.
• plaques produced by spleen cells from the buffer-treated control mice were large, rounded, and exhibited the expected clear and distinct zone of SRBC lysis.
• plaques produced by spleen cells from the sorbinil-treated mice were much smaller, more irregularly shaped, and less distinct. The most likely explanation for this observation is that the rate of antibody secretion by the sorbinil-treated spleen cells was less than that by the spleen cells from the control animals.
• Antibodies generated against the bacteriophage were quantitated by a phage inhibition assay, which is based on the observation that antibodies specific for the bacteriophage will inhibit the ability of the bacteriophage to lyse bacteria.
• Ochs, H.D., et al. Disorders of the B-cell system, in Immunologic Disorders in Infants and Children, E.R. Stiehm, et al., eds., W.B. Sanders, Co. Philadelphia, p. 239, 1980.
• Mice were treated daily for five days with sorbinil using the same dosage schedule as described in Experiment 6. Six hours after administration of
• mice were injected intravenously with 2 x 10 plaque forming units (PFU) of phage. Beginning with one week after this primary immunization mice were bled weekly, and serum samples from individual mice were tested for the ability to inhibit bacteriophage lysis of bacteria. The data from the first two weeks after the primary immunization are summarized as follows:
• sorbinil might inhibit the secretion of a soluble mediator of B lymphocyte maturation.
• the existence of two distinct types of soluble mediators that affect B lymphocyte maturation have been reported, one as affecting proliferation and the other as stimulating B lymphocyte maturation into plasma cells.
• Howard et al. have described a B cell growth factor found in induced supernatants of the mouse thymoma EL-4. Howard, M., et al., J. Exp. Med. 155:914, 1982. This factor is co-stimulating with anti-IgM antibodies in cultures of purified B cell cultures. This factor appears to stimulate B cell proliferation but not production of antibody forming cells.
• mice received sorbinil orally for one, two, or three days prior to sacrifice. The dosage was 11 mg/kg per day. Control mice received the buffer vehicle alone. Mice from these four groups were sacrificed and their spleens harvested. The spleens were teased into single-cell suspensions that were cultured with different concentrations of Con A for three or four days. One microcurie of tritiated thymidine was added to each culture 18 hours before the cultures were terminated. Incorporation of the tritiated thymidine into cellular DNA was measured by using a liquid scintillation counter. The results of this experiment are presented in FIGURES 20 and 21 and are summarized as follows:
• the dose response pattern to Con A was also similar in sorbinil-treated and buffer-treated mice, with 2 ⁇ gm/ml of Con A being most effective in cultures harvested on day three (FIGURE 20), and 5 ⁇ gm/ml being most stimulatory in cultures harvested on day four (FIGURE 21).
• mice receiving sorbinil prior to sacrifice exhibited a reduced proliferative response to the mitogen.
• FIGURE 20 the data relating to the cultures harvested on day three are summarized: The mice that received sorbinil for one or two days prior to sacrifice, had the most reduced proliferative response at 1 ⁇ mg of Con A/ml, the lowest dose of Con A employed: the proliferative response was reduced in those mice by 25% and 22%, respectively. At optimal doses of Con A (2 ⁇ mg/ml) the reduction was slightly less; 18% and 11%, respectively. Mice receiving sorbinil for three days prior to sacrifice exhibited the most reduced response to Con A stimulation.
• FIGURE 21 which represents the data obtained from cultures harvested on day four, when the proliferative response to Con A was declining, shows that the proliferative responses of spleen cells from mice treated with sorbinil for three days prior to harvest were reduced 25-58%. This reduction was greater than that observed in cultures from mice receiving sorbinil for two days prior to sacrifice: 5 to 18% reduction. Interestingly, in cultures of spleen cells from mice that had received sorbinil 24 hours prior to sacrifice, the proliferative response was enhanced by 3-30%. Discussion
• NSAIDs aldose reductase inhibitors that have the reactive conformation and inhibit DTH in mice do not alter normal arachidonic acid metabolism through lipooxegenase or cycloxygenase pathways. These are pathways inhibited by NSAIDs. As predicted, tolrestat and
• WF-3681 like sorbinil, inhibit selective aspects of the cellular and humoral immune systems by a completely new mechanism that may involve receptor sites for these ARI molecules.
• Mouse P338D cells were loaded with H-arachidonic acid and then stimulated with A23187, in the presence and absence of 10 -fi M/L sorbinil.
• the assay protocol was as described generally in Henderson, W.R., and S.J. Klebanoff, J. Biol. Chem. 258:13522-13527, 1983, which is hereby incorporated by reference. Sorbinil had no effect on the release of arachidonic acid metabolites from normal cells stimulated by A23187.
• PMNs Human polymorphonuclear leukocytes
• Thymocytes from animals that were administered sorbinil were tested q for incorporation of H-thymidine when stimulated by concanavalin A (Con A), a potent mitogen. 10 ⁇ g/Kg sorbinil was administered orally to mice 6 hours prior to q isolation of thymocytes. *H-thymidine incorporation in cells from sorbinil treated animals was nearly one-half that of cells from untreated animals.
• Human monocytes were isolated and tested for adherence in vitro in the presence and absence of sorbinil. Sorbinil concentrations as high as 10 — 6 M/L had no effect on the adherence of normal human monocytes in vitro.
• Experiment 17 Human macrophages were stimulated using LPS in vitro, and q
• popliteal lymph node assays were conducted on normal Balb C mice. Sorbinil had no effect on the swelling of popliteal lymph nodes in mice injeeted in the foot pad with cells from C57BL6 mice.
• Table has 4 columns, listing (left to right) each drug tested in column 1, drug concentrations in column 2, relative swelling in column 3, and effectiveness in column 4.
• NSAIDs quercitrin, acetaminophen, ibuprofen, and aspirin
• sorbinil, WF-3681, and tolrestat had nearly identical effectiveness on DTH (about 30%, at 6 ⁇ M).
• sorbinil was effective, as a single dose, when administered either before or after the antigenic challenge. Sorbinil, WF-3681, and tolrestat are chemically distinct reagents whose three-dimensional structures all contain the reactive conformation shown in

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