Patent Application: US-77466491-A

Abstract:
methods for treating and preventing late - phase allergic reactions with misoprostol in a pharmaceutically acceptable excipient are provided . tablet forms of misoprostol may be administered at doses of between 100 μg and 300 μg . the preparation may be administered alternatively as an aerosol . late - phase respiratory and cutaneous reactions to dust mites , food and occupational allergens , pollen , weeds , grass , drugs , animal dander and chemicals may be treated with the described misoprostol - containing agents . urticaria , contact dermatitis , asthma , allergic rhinitis and anaphylaxis are conditions which may be treated according to the presently disclosed methods and pharmaceutical agents .

Description:
the invention described herein demonstrates novel and particularly efficacious methods and agents for the treatment of late phase allergic reactions in a patient . the method provides relief from the painful symptoms typically attendant a late phase allergic reaction without the risk of a variety of side affects typically attendant the employ of currently available corticosteroid therapeutic treatments . misoprostol in tablet form ( 200 μg tablet ) was obtained as cytotec ® ( from g . d . searle and co ). anti ige was obtained boehringer scientific . dust mite was obtained from hollister - stier . codeine phosphate and histamine phosphate were obtained from sigma chemical . the following examples are presented only to describe preferred embodiments and utilities of the present invention , and to satisfy best mode requirements . the examples are not meant to limit the scope of the present invention unless specifically indicated otherwise in the claims appended hereto . the present example is presented to demonstrate the utility of using misoprostol in the treatment of histamine release - related allergic physiological responses in vivo . the present example also demonstrates the potential utility of the use of misoprostol with the claimed method and treatment in inhibiting release of allergic reaction mediators . the effect of misoprostol on histamine release from basophils was studied . histamine release was induced with anti - ige and mononuclear cell - derived histamine releasing factor ( hrf ). anti - ige was chosen because its mechanism of histamine release is similar to that of a standard allergen . hrf is a representative cytokine . leukocytes were isolated by sedimentation with 1 . 5 % hydroxyethyl starch in the presence of 0 . 01 m edta as an anticoagulant . the washed leukocytes were suspended in hacm ( 10 mm hepes buffer , ph 7 . 4 , containing 137 mm nacl , 5 mm kcl , 0 . 03 % human serum albumin , 2 mm cacl 2 and 1 mm mgcl 2 ). aliquots of 50 μl of leukocyte suspension were preincubated with various concentrations of misoprostol ( 10 - 8 to 10 - 4 m ) for 10 min at 37 ° c ., and then challenged with 50 μl of a predetermined concentration of anti - ige and hrf for an additional 45 min at 37 ° c . three sets of controls were run simultaneously : 1 ) cells preincubated with buffer and then challenged with secretagogues , 2 ) cells preincubated with misoprostol and then challenged with buffer , and 3 ) cells preincubated with buffer and challenged with buffer . each experiment was done in duplicate . at the conclusion of the incubation , supernatant was separated from the cells by centrifugation at 600 × g for 5 min at 4 ° c . and the histamine content of the supernatants were measured using an automated fluorometric analyzer . the total histamine content of the cells was measured by lysing the cells with 3 % perchloric acid . the percentage of histamine release was calculated according to the formula : ## equ1 ## in three experiments , misoprostol inhibited both anti - ige - and hrf - induced histamine release from leukocytes in a dose - dependent manner at high concentrations ( fig4 ). the maximal inhibition was observed at 10 - 4 m . in further studies , the inhibition of anti - ige - induced histamine release was carried out by preincubating basophils with misoprostol for different periods of time ( 5 , 10 , 15 , 30 min ) and at different temperatures ( 22 ° and 37 ° c .). the optimal preincubation time and temperature were found to be 10 min and 37 ° c . respectively . the present example is presented to demonstrate the activity of misoprostol for inhibiting the secretion of a major granular enzyme , beta glucuronidase , from an inflammatory cell type known as neutrophils . neutrophils are an important cell type involved in the inflammatory response . the results also demonstrate that misoprostol can inhibit neutrophils and / or the inflammatory response of neutrophils ( release of a granular enzyme ). the test was carried out according to schroeder et al . 23 briefly , leukocytes were separated from peripheral blood obtained from ten donors by sedimentation with hydroxyethyl starch . the buffy coat was washed and processed in hacm buffer . aliquots of cells were sequentially incubated with various concentrations of misoprostol ( 10 - 8 to 10 - 4 m ) or buffer for 15 minutes and then with cytochalasin b ( 500 μg / ml ) for 10 minutes . the c5a ( 1 μg / ml ) was added and the incubation continued for 30 minutes at 37 ° c . the tubes were centrifuged and 100 μl of supernatants was transferred to wells of an elisa plate containing 100 μl of 0 . 01 m p - nitrophenyl - b - d - glucuronide in 0 . 1 m sodium acetate , ph 4 . the elisa plate was incubated 18 hours at 37 ° c . 200 μl of 0 . 4 m glycine buffer , ph 10 , was added at the conclusion , and the plate read by an elisa reader at 546 nm . the results will be expressed as percentage release of beta - glucuronidase by c5a in the presence of misoprostol with reference to the release in the presence of buffer . leukocytes were thus preincubated with various concentrations of misoprostol and then challenged with c5a . the released beta - glucuronidase was assayed by its ability to metabolize p - nitrophenyl - b - d - glucuronide . in three experiments , c5a induced the release of beta - glucuronidase from neutrophils in a dose dependent manner , and the optimal concentration for release was 1 μg / ml . the mean beta - glucuronidase release was 19 ± 3 %. misoprostol was used in one experiment , and it inhibited c5a - induced enzyme release in a dose - dependent manner . the maximal inhibition was 48 % at 10 - 5 m concentration . the present study demonstrates the effectiveness of misoprostol in a method for treating and preventing late - phase allergic reactions , most particularly late - phase allergic cutaneous reactions . misoprostol was administered as an oral tablet . the serum level of misoprostol after an oral dose of 200 μg was found to be 300 - 600 pg / ml (˜ 1 - 2 × 10 - 9 m ). while the half life of misoprostol is between about 1 . 5 to 1 . 7 hours , the present study demonstrated effective inhibition of late - phase cutaneous allergic reaction to dust mite antigen 4 - 5 hours after exposure . this indicated to the inventors that the mechanism of action of misoprostol is different from the mere inhibition of histamine release from basophils . the inventors &# 39 ; observation that misoprostol does not affect immediate allergic reactions ( i . e . &# 34 ; early - phase &# 34 ;), also indicates that misoprostol does not function simply to inhibit histamine release from basophils . it is postulated that misoprostol is acting at a very early phase of inflammation , since it is eliminated from the body long before the peak of a late - phase allergic reaction . the same pharmacological activity is expected by the present inventors from topical application of misoprostol , such as , for example , when administered as a cream or ointment in association with liposomes . six patients with known skin test allergic reactivity to dust mite ( dermatophagoides pteronyssinus / farinae ) were studied . all patients had an initial intradermal testing with the allergen to determine the dose that evoked a late - phase cutaneous response . patients were asked to withhold medications . patients were required to follow the following guidelines in the use of drugs that are known to modulate skin reactivity : short - acting antihistamines must be stopped for 72 hours , and tricyclic antidepressants and benzodiaxepines for two weeks . patients on astemizole and systemic steroid were excluded . any patient with a history of recent narcotic drug intake or having taken an investigational drug within one month of the study was excluded . other exclusion criteria include skin infections or skin disorders , history of anaphylaxis , and history of systemic or moderate - severe localized reactions to histamine , codeine , or allergen extract , and patients with systemic illnesses requiring uninterrupted medication . after an overnight fast , patients had intradermal skin testing with dust mite ( 50 au / ml ), codeine phosphate ( 1 mg / ml ), histamine phosphate ( 0 . 275 ), and a saline control . 50 au / ml of dust mite had previously been determined by the inventors to induce a late - phase skin reaction in most allergic donors . the longest and perpendicular diameter of the wheal response were read at 15 minutes . the measurement was repeated hourly for 5 hours . previous experience of the inventors indicated that the peak of the late - phase skin reaction occurred at 4 - 5 hours following the immediate (&# 34 ; early - phase &# 34 ;) reaction . three days after an initial treatment , each patient was given a 200 mcg tablet of misoprostol ( cytotec ®; g . d . searle & amp ; co .) after an overnight fast . thirty minutes later ( time of peak blood level following an oral dose in a fasting subject ), skin testing was performed as described above . the immediate and late - phase reactions were measured as above . the allergic reaction to skin testing is presented as the mean of the longest and the perpendicular diameters . all six patients developed an immediate allergic reaction at 15 min ( fig1 ). the wheal response to histamine ( fig2 ) and codeine ( fig3 ) subsided after the immediate phase , although the codeine - induced response cleared more slowly . in contrast , the reaction to dust mite continued to increase in intensity and reached its plateau at 4 hours . administration of misoprostol did not affect the immediate phase allergic reaction , but it significantly prevented the protracted late - phase reaction . there was no effect of misoprostol on codeine - and histamine - induced immediate allergic reaction . misoprostol and cutaneous late - phase allergic reaction double blind placebo control study the present example presents clinical data collected from 12 adult males and females . females who participated in the study were reproductively incompetent , as they either had undergone a tubal ligation or hysterectomy . twelve patients with known skin test reactivity to dust mite ( dermatophagoides pteronyssinus / farinae ) were studied . all patients had an initial intradermal testing with the allergen to determine the dose that evokes a late cutaneous response . patients were asked to withhold medications . the following guidelines were maintained for drugs that are known to modulate skin reactivity : short - acting antihistamines must be stopped for 72 hours , and tricyclic antidepressants and benzodiazepines for two weeks . patients on astemizole and systemic steroid were excluded . any subject with a history of recent narcotic drug intake or having taken an investigational drug within one month of the study was to be excluded . other exclusion criteria included skin infections or skin disorders , history of anaphylaxis , and history of systemic or moderate - severe localized reactions to histamine , codeine , or allergen extract , and patients with systemic illnesses requiring uninterrupted medication . the study was conducted in a double - blind placebo - controlled manner . placebo and misoprostol ( 200 μg ) were put in look - alike capsules . the capsules were stored by one of the investigators who did not see the patients . on the study day , the coded capsule containing the placebo or the active drug was given to the nurse in a blinded fashion . the coded capsule was then administered to the patient . the patient was also blinded as the content of the capsule . the skin testing procedure was conducted 30 minutes later . the procedure was repeated 5 days later . each patient received either placebo on 1 day or misoprostol on another day . each patient had intradermal skin testing with dust mite ( 50 au / ml ), codeine phosphate ( 1 mg / ml ), histamine phosphate ( 0 . 275 mg / ml ), and a saline control . the present inventors had previously determined that 50 au / ml of dust mite induced a late - phase skin reaction in most allergic donors . the longest and perpendicular diameter of the wheal response were read at 15 minutes . the measurement was repeated at 1 hour and hourly for 5 hours . previous experience of the inventors indicated that the peak of the late - phase skin allergic reaction occurred at 4 - 5 hours following the immediate reaction . the allergic reaction to skin testing is presented as the mean of the longest and the perpendicular diameters . all twelve patients developed an immediate allergic reaction at 15 minutes ( fig5 ). the reaction to dust mite continued to increase in intensity and reached its plateau at 4 hours on the placebo day . administration of misoprostol did not affect the immediate phase allergic reaction , but it significantly ( p & lt ; 0 . 05 ) inhibited the protracted late - phase reaction . there was no effect of misoprostol on histamine - and codeine - induced allergic reactions ( fig6 and 7 , respectively ). the present example is provided to demonstrate the utility of misoprostol in the treatment of nasal immediate and late allergic reactions in humans . the effect of misoprostol on nasal immediate and late allergic reactions will be investigated on 20 patients in a double - blind placebo - controlled randomized study . misoprostol or placebo ( lactose ) will be administered in look - alike coded capsules 30 minutes before the nasal challenge . the same coded drug will be given again 6 hours later . symptom score will be monitored every 30 minutes until the immediate reaction , and then hourly for 12 hours . the second challenge will be conducted within 5 - 7 days . nasal challenges will be conducted at the same time of the day to avoid changes in the bioresponse due to the diurnal rhythm . nasal antigen challenges will be performed on allergic patients as described by naclerio et al . initially patients will be challenged with the diluent using a spray that delivers 0 . 2 ml each squirt . symptoms will be monitored for 30 minutes . then 0 . 2 ml of a predetermined dose of the appropriate allergen will be delivered into each nostril at increasing doses at 30 minutes intervals , and symptoms monitored . patients will be evaluated for their response with reference to a symptom score well known to those of skill in the art . patients will be evaluated for their response with reference to a symptom score , well known to those of skill in the art . the symptom score for three cardinal symptoms is calculated the following way : sneezes 0 ( none ), 1 ( 1 - 8 sneezes / hr ), 2 ( 9 - 16 / hr ), and 3 (& gt ; 16 / hr ); rhinorrhoea and nasal congestion are graded as 0 ( none ), 1 ( mild ), 2 ( moderate ) and 3 ( severe ). the maximal possible score is 9 . each patient will be asked to record the symptom score hourly for 12 hours on a diary card . if the symptoms score reaches 6 at any point during the challenge , the next concentration of the allergen will be omitted . however , if the symptom score is between 4 and 6 , half of the next concentration will be applied . with the results obtained from the above described study , effective doses of nasally applied misoprostol may be defined for clinical use in humans . the present example is provided to outline the proposed utility of misoprostol in the treatment of respiratory late - phase allergic reactions in humans . such respiratory late - phase allergic reactions are involved in , for example , asthma and allergic rhinitis . the effect of misoprostol on allergic bronchial asthma can be studied using the existing formulation as , for example , using a 200 mcg tablet of misoprostol such as that obtainable from cytotec ®( g . d . searle , inc .). the dosing regimen is 1 tablet every 6 hours . the clinical efficacy of the drug can be studied in a number of ways , including the measurement of reduction in daily symptom score ( shortness of breath , wheezing , and cough ), reduction in the usage of bronchodilators ( albuterol inhaler or others ) and improvement in peak expiratory flow rates and other spirometric indices as well as in bronchial hyperreactivity . another possibility is the intrabronchial delivery of the drug in lipid emulsions as an aerosol . misoprostol will be dissolved in soybean oil ( 200 μg / ml ) and then incorporated in a lipid emulsion ( final concentration 20 μg / ml ). this emulsion will be aerosolized using a standard nebulizer . the drug will be delivered at a dose of 3 - 5 μg / minutes for 5 minutes . patients will be treated with the aerosol treatment every 6 hours . the clinical efficacy will be monitored as described above . still another possibility would be to administer the misoprostol as a nasal aerosol , for example in the treatment of allergic rhinitis , as described in example 5 . twenty allergic asthmatic patients will be recruited for this double - blind placebo - controlled study . allergen challenge will be conducted on two separate days one week apart . the inclusion criteria include 80 % of the previously best recorded fev1 and fvc on the day of the study . patients will have a baseline spirometry . qualifying patients will receive misoprostol or placebo in coded capsules in a randomized fashion . thirty minutes later the patient will undergo allergen challenge test . after an initial saline inhalation test , the patient will be challenged with a predetermined dose of a skin test - positive allergen extract . spirometry will be repeated at 5 , 10 , 15 , 30 min , 1 hour and then hourly for 10 hours . all patients will receive another dose of the same coded capsule 6 hours after the initial dose . the study will be repeated one week later . bronchial challenges will be conducted at the same time of the day to avoid changes in the bioresponse due to the diurnal rhythm . patients will have a baseline spirometry in the morning of the study day and then inhale 5 breathes of saline from a devilbiss 646 nebulizer connected to a rosenthal - french nebulization dosimeter , both apparatus being well known to those of skill in the art . compressed air is passed through the dosimeter for nebulization . spirometry is repeated at 5 and 15 minutes . if no significant drop in fev1 ( 20 % fall is considered significant ) is noticed , patient will proceed to have allergen challenge with a predetermined dose of a skin test positive allergen . the same nebulizer system will be used for the allergen challenge . patients will be allowed to take inhaled bronchodilators if they experience even only moderate respiratory distress . for severe respiratory distress , 0 . 3 ml of epinephrine sc , bronchodilators via nebulizer and theophylline iv will be started and the study discontinued . topical administration of misoprostol into the nose and lungs may also be considered . this may include aerosolization of the drug through a metered dose inhaler or through a nebulizer . the concentration of allergen administered to the patient will be increased until the fev1 ( forced expiratory volume in 1 second ) falls 20 %. the results of in vitro study will be analyzed for statistical significance of difference between samples treated with misoprostol and with placebo or buffer in each group . the codes for the blinded drugs for in vivo studies will be deciphered after completion . the results of cutaneous ( mean wheal diameter ), nasal ( symptom score ) and bronchial response ( fev1 ) from the misoprostol day will be compared with that from the placebo day . the statistical significance of differences will be analyzed using wilcoxon &# 39 ; s rank sum test or signed - rank test . all statistical analyses will be considered significant at p & lt ; 0 . 05 . the following references are specifically incorporated herein by reference in pertinent part for the purposes indicated . 1 . bauer , rf ( 1985 ), digestive diseases and sciences , 30 ( 11 ): 118s - 125s . 2 . feldman , m ( 1990 ) the american journal of medical sciences , 300 ( 2 ): 116 - 132 . 4 . goodman and gilman , eds ., the pharmacological basis of therapeutics , 18th edition ( 1990 ), pp . 574 - 588 , pp . 610 - 611 , pp . 618 - 619 , and p . 638 . 5 . merck manual ( 1987 ) 14th ed . r . berkowetal ., eds ., pp 304 - 306 . 6 . ham ea , soderman dd , zanetti me , et al ., ( 1983 ) proc . natl . acad . sci . usa , 60 : 4349 . 7 . marone g , kagey - sobotka a , lichtenstein lm ( 1979 ), j . immunol , 123 : 1669 . 8 . kunkel sl , chensue sw ( 1985 ), biochem . biophys , res . commun ., 128 : 892 . 9 . g . d . searle investigational brochure &# 34 ; arthritis prostaglandins research challenge &# 34 ; page iv - 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