CA1320905C - Treatment of immuno-resistant disease - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Neoplastic disease, hyperallergenicity, autoimmune disorders characterized by chronic tissue degenerative inflammation and immuno-resistant viral infections are treated by the administration of iterferon at a dosage of about 0.1 to about 5 IU/lb per day by contacting said interferon with oral/pharyngeal mucosa. Interferon is administered in solution or in a novel solid unitary dosage form adapted to be dissolved in saliva when placed in the mouth.

Description

1 320qO5 TREATMENT OF IMMUNO-RESISTANT DISEASE

~AÇg~R~p QF THE I~VE~IO~

This invention relates generally to an improved method of treating disea~es of immuno-pathologic etiology in warm-blooded vertebrates us;ng interferon in low oral dosages. This in~ention also relates to the use of interferon in low oral ~osages to potentiate disease-corrective immune responses in warm-blooded vertebrates afflicted with immuno-resistant diseases charasterized by apparent hyperactive or hypoactive immune system function.
rInterferon~ is a term generically comp~ehending a group of vertebrate glycoproteins and proteins which are known to have various bioloqical activities, such as antiviral~ antiproliferative, and immunomodulatory activity at least in the species of animal from which such substances are derived. The following definition for interferon has been accepted by an international committee assembl~d to devise a system for the orderly nomenclature of interferons: ~To qualify as an interferon a factor must be a protein which e~erts virus nonspeciic, antiviral activity at least in homologous cells through cellular metabolic processes involving synthesis of both RNA and protein."
Journ~l o Inter~eron Resear~h, 1, pp. vi (1980).
~Interferon~ as used herein in descr;bing the present invention shall be deemed to have that definition.
Since the first descriptions of interferon by Isaacs and Lindeman [See, Pro~ç. Roy. SQc. LQndon (Ser.
~, Yol. 147, pp. 25~ e~ se~. (1957) and U.S. Patent No.

, .

3,699,222], interferon has bee~ the subject of intensive research on a worldwid~ basis. The literature is replete w;th publications concerning the synthesis of interferun, its proposed molecular characterizations, its clin;cal applications and proposed mechanisms of its antitumor, antiviral, and immune ~ystem activities.
Because of the intensity and disparate origins of research concerning interferon and its characteristics and uses~ there e~ists a substantial lack of unifoxmity in such matters as classification of anterferon types. There are also numerous, sometimes contradictory, theories concerning the mode of action of interferon in producing clinical effects.
Although originally isolated from cells of avian origin (chick allantoic cells), interferon production has been observed in cells of all classes of vertebrates, including mammal~, amphibians, birds and reptiles. Interferon production by vertebrate cells is seldom spontaneous but is often readily Uinduced~ by treatment of cells (in vivo or in vi~rg) with a variety of substances including virl~ses, nucleic acids~
(including those of viral origin as well as synthetic polynucl~otides), lipopolysaccharides, and various antigens and mitogens.
Interferons have generally been named in terms of the species of animal cells producing the substance (e.g.~ human, murine, or bovine3, the type of cell involved Se.g., leukocyte, lymphoblastoid, fibroblast) and, occasionally, the type of inducin~ material responsible for interferon production ~e.g~, virus, .- 1 320905 immune). Interferon has been loosely classified by some researchers according to induction mode as ei~her Type I
or ~ype II, with the former cla~ification comprehending viral and nucleic acid induced i~terferon and the latter clas~ including the material pro~uc~d ~s a lymphokine through induction by antigens and mitogens. More recently, the international committe~ ~evising an orderly nomenclatur~ ~ystem for interferon has classified interferon into type~ on the basis of antig~nic specificities. In this newer classification, the designations alpha (a), beta (B), and gamma (y) have been used to correspond to previous designations of leu~ocyte, fibroblast~ and type II (immune3 interf~rons, respectively. Alpha and beta interferons are usually acid-stable and correspond to wha~ have been called type I interferons; gamma interferons are usually acid-stable and correspond to what has been called type II
interferons. The international committee's nomen~lature recommendat;ons apply only to human and murine interferons. ~ournal of Int~rferon Re~earch, 1 pp. vi (1980).
In its earliest applications, interferon was employed e~clusively as an antiviral agent and the most successful clinical therapeutic applications to date have been in the treatment of viral or virus-related disease states. It became apparent, however, that e~ogenous interferon was sometimes capable of effecting regression or remission of various metastatic diseases.
An overv.iew of current clinical trials of interferon as an antiviral an~ antiprolierative therapeutic a~ent is 1 320qO5 contained in In~Q~ç~Qn: In Yiv~ linical Studi~, Volume 4, Eds: N. B. Finter and R. R~ Oldham, Academic Press, ~ew York, 1985.
~he clinical agen~ o~ choice for the presen~
inven~ion is human leukocyte inter~eron, ~ma~s-produced~
by procedures involving coll~ction and purification of vast quantities of human bufy coat leukocy~es, ;nduction w;th virus, and isolation from culture media.
In the work described above, interferon has been administered parenterally, i.e., intramuscularly and intradermally, with ~om successful topical and intranasal usages having been reported. It has seldom been administered intravenQusly because of substantial adverse effects attributable to ~contaminantsW in crude and even hi~hly purified isolates.
As discussed above, there has been a ~ignificant research effort directed to the evaluation of therapeutic effects of intereron for a wide variety of diseases having an auto-immuno-pathologic basis.
Before applicant's first report of successful oral administration of interferon in his U.S. Patent Application Ser;al No. 415,525 (now U.S. Patent 4~462,985), there was no recognition in the art of the potential offered by oral administration of interferon.
The generally held belief was that interferon could not survive the di~esti~e conditions of the upper ~limentary canal.
Since applicant's first disclosure of the immunotherapeutic benefit achievable via oral administration of interferon of heterologous mammalian ~5--species, he has continued to investigate the efficacy of orally administered interferon. In U.S. Patent No.
4,497,7g5, issued February 5, 1985, applicant deseribed and claimed the use of interferon admin;stered orally or via intravenous administration to stimulate appetite and eed e~ficiency of bovine and porcine species.
Human alpha-interferon has been marketed under the trademark Agriferon~ by Immunomodulator Laboratories, Inc. (~IML~) o~ S~afford, Te~as for veterinary use in Te~as since February 1985. The product is sold for oral administration to cattle to promote growth and feed efficiency and to prevent or treat viral respiratory in~ections. IML began selling an alpha-interferon product for horses in 198~. Both produc~s are sold under a license of my U.S. Patent 4,462,985.

SUMMARY OF THE INVENTIQN
Interferon ~ontacting the oral and/or pharyngeal mucosa, i~ amounts of less than 5 IU/lb of body weight per day is consistently effective to potentiate disease-corrective immune responses in ~?

vertebrates afflicted with immuno-resistant ~iseas~
states characterized by apparent hyperac~ive or hypoactive immune sy~tem function. Trea~ment in accordance with the present invention has been shown to effect remission of neoplastic disease, hyperallergenicity, immuno-resistant or immuno-debilitating viral infections and autoimmune disorders characterized by chronic tissue degenerative inflammation.
I

pE~ILED D~RIP~IQN OF THE I~VENTIQ~

The clinical agent of choice for use in the present invention is human leukocyte interfsron (human alpha-interferon), ~mass-produced~ by procedures involving collection and purification of quantities of human buffy coat leukocytes, induction of intereron production with virus9 and isolation of culture media.
(See ~Preparation of Human Alpha-Intereron" below.
Also acceptable for use in accordance with present intention are human alpha-interferon products ~produced by recombinant DNA technology and now commercially available from Schering-Plough ~as Intron0) and Hoffmann-LaRoche tas Roferon~) and approYed by the FDA
for treatment ~parenterally~ of hairy cell leukemia of man. Such recombinant interferon products are believPd to be particularly effectiYe when used in combination.
Gamma interferon is also available by recombinant technology and is presently undergoing clinical trials .: .

1 320')05 by Genentech and others. Fibroblast interferon ~beta-interferon) can be prepared in accordance with E~ample 1 in applicant's U.S. Pat~nt No. 9,462,985 issued ~uly 31, 19~4.
~ nterferon of hum~n and murine origins has been quantified in the art in terms of International Uni~s ~IUa). As used here;n, a "unita of interferon (to be distinguished from ~IU~ shall mean the reciprocal of a dilution o intereron-con$aining material that, as determined by assay, inhibits one-half the number of plagues of a ~hallenge virus, the challenge virus being the vesicular stomatitis ~irus (UVSVn). So quantified a ~unit" of interferon is routinely found to be about one-tenth the quantity of interferon represented by one ~IU.~ In other words, for thP purpose of defining the present invention, 1 unit ~0.1 IUo ~he present invention relates to an improved . mPthod of treatment of immuno-resistan~ disease states with interferon. The present invention is directed to ~he treatment of diseases in warm-bloo~ed vertebrates, particularly cestain diseases which the immune system of many species is poorly eguipped to handle, as evidenced by either a lack of disease defeating response and/or an apparently misdirected immune response resulting in a chronic tissue degenerative in1ammatory condition or other physical complications. Wh;le there has been a signiicant resear~h effort directed to the u~e of interferon for treatment of such diseases, reported results, although positive overall, have been ~' 1 320qos i~consistent, The principle reason for ~uch inconsistency in view of my most recent research efforts is that earli~r investigatoræ have failea to d~fine optimum dosage and rout2 of interferon administra~ion.
The present invention is based on applicant~s dis~o~ery that interferon san be used as a consistently efective therapeutic agent for treatment o diseases having an immunopathologic basis - characteriz~d by inadequate immune response an~ persistence of the disease or by an apparent hyperactive immune response resulting in tis~ue deqenerative inflammatory conditions and related physical manifestations. Applicant has found that interferon, contacting the oral and pharyngeal mucosa in amounts from about 0.01 to about 5 IU/lb of body weight per day, is consistently efficacious for the treatment of disea~es to which the t immune system of many warm-blooded Yertebrates does not effectively respond.
Disease conditions treated in accordance with the present invention include apparent autoimmune disorders characterized by a chronic tissue degenerative inflammatory condition. Diseases so characterized include multipl4 sclerosis, rheumatoid arthritis, stomatitis, and lupus erythematosus. Treatment of such disease is in accordance with the present invention comprises administering interferon at a dosage of 0.01 to about 5 IU/lb per day in a dosage form adapted to promote contact of said dosage of intsrferon with the oral and pharyngeal mucosa of said animal. Preferably, , 1 3~0905 g the ~05age of ~nterfsron ls rom 0.1 to ~bout 4.0 IU/lb per ~ay~ more preferahly O.S to about ~.5 IU~lb of body weight per day. Alpha ~n~erferon, derived from tissue culture or hy recombinant DNA t~chniques~ is ~ preferred therapeutic agent in accoraance with this invention.
Some data have indica~ed bet~er e~ficacy, i.e., a more pronounced immunomodulatory effect, where the int~rferon ;s not homologous to ~he ~pecies being treated. Alpha interferon can be administered alone or in combination with beta interferon or gamma in~erferon.
It is c~itical that the interferon be administered in a dos~ge form adapted to assure maximum contact of the interferon in said dosage form with the 1~ oral and pharyngeal mucosa of the human or animal undergoing treatment~ Contact of interferon with the mucosa can be enhanced by ma~imizing residence time of the treatment solution in the oral or pharyngeal cavity. Thus, best results ~eem to be achieved in human patients when the patient is requested to hold said solution of interferon in the mouth for a period of time. Contact of interferon with the oral and pharyngeal mucosa and thereafter with the lymphatic system of the Sreated human or animal is unquestionably the most efficient method administering immunotherapeutic amounts of interferon.
Another disease condition responding to treatment in accordance with the present invention is neoplastic diseaseO Thus, the administration of interferon in accordance with the above description can, alone or in combination with other drugs or therapy, help effect remi~siQn o canc~rs such as maliqnan~
lymphoma, melanoma, mesothel~oma, ~lrkîtt lymphoma and na~opharynqeal carcinoma and oth~r neoplas~ic diseases, 2specially thos~ of known or suspected viral etiology.
Based on the results ob~rved to date, it is beli~ved that applicant's presently described method of treatment will ~imilarly help effect remission of Hodgkin's Disease and leuk~mia~
Other disease ~onditions responding to treatment in accordance with the present invention are infectious diseases of viral origin in, for e~ample, human, aYian, porcine, canine and feline species.
Significantly, viral infection typically e~hibiting persistent resistance to treatment have shown a dramatic response to ~reatment with interferon in low doses contacting the oral and pharyngeal mucosa of infected patients. B~neficial results have bPen attained utiliziDg the present m~thod to treat dogs having canine parvovirus and canine herp~svirus infections. Further, felins leukemia and feline infectious peritoni~is have heen shown ts be particularly susceptible to treatment with alpha interferon and beta interferon in accordance with this invention.
E~emplary of human viral infections showing r~markable response to treatment in accordance with the present invention are infections of human rhinovirus (common cold), herpe~ sim~le~ I virus ~cold sores) and 3~ human papovavirus (warts). Based on treatment results 1 320~05 to date, it is espected that contact of interferon at low do~age with the oral and pharyngeal muco~a will provide an ef~scti~e treatment for Acguired Immune 5 Deficiency ~yn~rome (AIDS3 a~d disease con~itions having the herpes ~imple~ iru as the causatiYe agent. A
patient e~periencing a condit;on of virsl myocarditis has r~sponded fe~orably to the present treatment. Warts often dissipate within 6i~ to eight weeks after initiating treatment in accordance with this invention.
Interferon administration in accordance with this invention can also be used to h~lp pre~ent viral infections, fsr e~ample, infections by the causitive agents of flus and colds, and to minimiæe the symptoms associated with such viral infections.
Other afflictions responding to contact of low dosage interferon are hyperallergenic conditions such as asthma. One ~side effect~ noted by patients ~reated in accordance with this invention is improved skin comple~ion. Thus, administration of interferon in dosages of about 0.01 to a~out 5 IU/lb of body weiyht per day is effective to treat acne, specifical~ly and improve human ~kin comple~ion generally.
Further, stimulating the immune system by oral contact with low dosage interferon i~ believed to assist the body in fighting bacterial infection. Treatment in accordance with this invention alone or in combination with therapeutic amounts of antibiotics can be especially ef ctive in knocking down infections of antibiotic resistant microorganisms.

' 1 320q~5 Administration of interferon in ~ccordance with the present inv~ntion is preferably continued until the symptoms of the disea~e condition being treated t subside. This can range from a p~riod of one day, for e~ample, where a human rhinovirus is the disease causative agent, to a period cf up to sis months for treatment of neoplastic disease. Rheumatoid arthritis patients are pain free within 2 to 10 days of init;ating treatment in a~cordance with the present invention.
However, treatment of that disease is preferably conducted by administration of interferon for up to about three (3) months.
~sily dosage of interferon can be administered as a single dosage or, preferably, it is divided and administered in a multiple-dose daily regimen. A
~taggered regimen of at least one, for e~ample, one to three days treatment per week or month, can be used as an alternative to continuous daily treatment.
2~ Interferon can be administered in accordance with this invention in e;ther a liquid (solution) or solid dosage form. Thus interferon can be administered dissolved in a buffered aqueous solution typically containing a stabilizing amount ~1 5% by weight) of 2~ blood serums. E3emplary of a buffered solution suitable as a carrier of interferon administered in accordance with this invention is phosphate buf ered saline prepared as follows:
A concentrated (20s) solution of phosphate 3~ buffered saline (PBS) was prepared by dissolving the 1 320qos following reagent~ ;n sufficient water to make l,oOo ml of solution: sodium chloride, 160 grams; potassium chloride, ~.0 ~rams; sodium hydrogen phosphat~, 23 grams, potassium dihydrogen phosphate, ~.0 grams; and optionally phenol red powder, 0.4 grams. The solution is ster~lized by autoclaving at 15 pounds pressure for 15 minutes and then diluted with additional water to a ~ingle strength concentration prior to use.
1~ - Alternatively the interferon can be formulated into f lavored or unflavored solutions or syrups using a buffered aqueous 601ution of interferon as a base with added caloric or non-caloric sweeteners, flavor oils and pharmaceutically acceptable surfactant/dispersants.
1~ It is also contemplated by the present invention to provide interferon in a solid dosage form such as a lozenge adapted to be dissolved upon contact with saliva in the mouth with or without the assistance of chewing. Such a unitary dosage form is formulated to release about 1 to about 1500 IU of interferon upon dissolution in th~ mouth for contact with the oral and pharyngeal mucosa. Thus a un~tary dosage for~ of interferon in accordance with this invention can be prepared by art-recognized techniques for forming 2S compressed tablets ~uch a~ chewable vitamins.
Similarly, interferon can be incorporated into starch-based gel formulations to form a lozenge which will dissolve and release interferon for contact with the oral mucosa when held in the mouth. Solid unitary dosage forms of interferon for use in accordance with ~14-the present i~vention can be prepared utîlizing art recognized ~o~age formulation techniques. The pH of ~uch formulatio~ can range from about 4 to abou~ 8.5.
0 course, in proce~si~g to ~uch unitary dosage forms on2 should avoid heating a pre-do~age form formulation, after addition of interferon, above about 50 Centigrade. E~emplary of a solid dosage form for animal use is a molasses block ~ontaining effective amounts of interferon. I
P~çparation Qf Human Alph~-ln~erf~r~n Human alpha-interferon can be prepared through the following procedure, commonly referred to as the Cantell procedure. The process begins with packs of human leukocytes, ohtained ~n this case from the Gulf Coast ~egional Blood Center, Houston, Te~as. The buffy coats in these packs are pooled into centrifuge bottles, and then are diluted with 0.83% ammonium chlorideO The mi~ture is incubated for 15 minutes with intermittent shaking, and is then centrifuged for 20 minutes at 2000 rpm. The supernatant is discard~d, and the cell pellets are resuspended with a minimal volume of sterile phosphate buffered saline (PBS). The mi~ture is then diluted with ammonium chloride and centrifuged. The supernatant is ayain discarded, and the remaining cell pellets are resuspended with a minimal volume of a tissue culture medium such as Minimal Essential Medium (MEM), available from KC Biological. The cell concentration is determined with a Coulter counter.

, - Interferon induction takes place in glass or plastic ~ottles. The i~duction medium contains MEM, 75mM ~epes (available from Calbiochem), 75mM Tricine (available from Sîgma Chemical Co.), human agamma serum (18mg~ml), and gentamycin sulfate (from MoA~
Bioproducts; 50mcg/ml). The c~lls are adde~ to ~h~
induction vessels at a final concentration of about 5 to 10 million cells per milliliter. The induction vessel is incubated in a 37~C water bath, and interf~ron alpha is added as a primer. I
After two hours, Sendai virus is added to the induction mi~ture. This causes alpha interferon to be produced in the supernatant by the leukocytes. After a 12-18 hour incubation time, the induction mi~ture is centrifuged. The cells are discarded~ and the supernatant is then purified.
The crud~ interferon is chilled to 10C or below in an ice bath. Five molar potassium thiocyanate is added to obtain a final concentration of 0.5M. This solution is stirred for 15 minutes, and then its pH is lower~d to 3.3 by adding hydrochloric acid. The mi~ture is then centrifuged at 2800 rpm for 30 minutes, and the supernatant is discarded.
The pellets are then resuspended in 95% ethanol and are stirred for 15 minutes. This suspe~sion is centrifuged at 2B00 rpm for 20 minutes, and the pellets are discarded. The pH of the supernatant is then adjusted to 5.8 with sodium hydroxide. The mi~ture is stirred for 10 minutes, and then centrifuged at 2800 rpm .

1 320qO5 for 20 minut~s. The pellets are discarde~. The pH of the aupernatant is th~n a~justed to 8 with ~odium hydro~id~. Thi~ &olution is stirred for 10 minutes, followed ~y centrifuga~ion at 2800 rpm for 20 minutes.
The supernatant is di~carded, and the pellets ~re resuspended with 0.5~ potassium thiocyanate in a 0.1~
~odium phosphate buffer. This suspension is stirred at 4C.
Ne2t, the su~pension i~ centrifuged at 2800 rpm ~or 20 minutes, and the pellets are discarded. The pH
of the supernatant is adjusted to 5.3 with hy~rochloric acid. After stirring or 10 minutes and centrifugation, the pH of the supernatant is adjusted to 2.8 with hydrochloric acid, followed by further stirring for 2~
minutes. This mi:~ture is centrifuged at 28DO rpm, and the resulting pellet is purified human alpha-interferon.
The pellet is resuspended with 0.5M potassium thiocyanate in O.lM sodium phosphate buffer, having a pH
of 8Ø It is then dialyzed against PBS at 4C, with two changes of PBS. This mixture is then centrifuged and the precipitate is discarded. The remaining pusified alpha interferon is sterilized by filtration through a 0.2 micron filter. A human alpha-interferon is produced in accordance with this procedure by Immuno Modulators Laboratories, Inc., Stafford, Te~as, and sold under the trademark Agriferon~ for use in cattle and Equiferon~ for use in horses.
Other procedures known to those skilled in the art are available for making interferons, such as human alpha-interferon a~d human gamma-interf~ron. For e~ample, U.~. Patent~ ~,37~,~21 and ~,4600685 disclose methods o~ making human qam~a-inter~eron. ~ method of making bovine fibroblas~ ~beta) in~erferon i~ disclosed in applicant's U.~. patent 4,462,985.

~linical Studies Tables 1-4 below ~ummarize the results of clinical studies of the administration of interf~ron by veterinarians orally to 137 dogs and cats as of ~ovember, 1985. The studies were conduc~ed with both human alpha-interferon and bovine beta-interferon.
Table~ 1-4 compare survival rates of pets with feline leukemia virus-associated diseases or canine parYovirus disease. Unequal numb~rs of pets were treated with each type o~ interferon; bovine beta-interferon was given to 78 pets and human alpha-interferon was given to 59 pets.
Bovine beta-i~terferon was produced in flasks of confluent monolayers of bovine fetal kidney (BFK~
cells. Culture supernatant was Aarvested 24 hours after bluetongue virus induction of BFK cells~ The supernatant was dialyzed 24 hours in a p~ 2.0 buffer and for another 24 hours in a PBS ~pH 7.4) buffes before interfexon assay. Procedures for the assay and characterization of bovine beta-interferon were essentially as described by Rosenquist and Loan, Am~ri~an JQ~rnal ~ Ve~erinary Res~ch, 28; 619-628, 1967. Interferon titers as ~units~ were expressed as , the reciprocals of the'dilutions that provided a 50%
reduction in the number of VSV plaques as ~ompared with the number in control cultures. The BFR cell culture interferon produced by this method had an average titer of 7,000 unit~ per milliliter. Dogs were given bovine beta-interferon, 5-10 ml/dos~, as least thre~ times/day after a diagno~is of CPV di ease. Cats positive by ELISA for feline leukemia virus and e~hibiting clinical signs of disease wer~ given 1 ml~10 lb of body weight 2-3 times daily for five days. After a five day interval, cats were retreated at least once for another five days.
Human alpha interferon was obtained from IML, Inc. of Houston, Te~as. Cases were treated with lot AO26 applied at 6 X 106 IU/ml. Lot ~G26 of human alpha interferon was diluted 1:150 i~ Eagles' minimum essential medium ~MEM~ and used as the stock solution from which 1 ml was further diluted 1:1000 with 1 liter 2~ of MEM for treatment. The usual dose of human alpha interferon was 4 IUflb body w~ight given at least three times daily after a diagnosis of CPV disease w~s made.
For feline leukemia, cats were treated with human alpha-interferon 2-3 times daily for five days as reported for bovine beta interferon.
Significantly tPC0.05~ more cats lived si~ and twelve months after diagnosis and treatment for feline leukemia virus if alpha-interferon was given, compared to treatment with bovine beta-intereron. Significantly (P<0.05) more dogs survived CPV disease when given alpha interferon (92%) compared ~o those ~ogs given bo~ine b~ta-interferon (69%~.

Summary o Survival Date from clinically ill cats positive for FeLV.
_ _ Months Af~er T~a~ment 1~ Treatment _ 1 6 12 Human alpha-IFN 25~33 21/32~ l9J31 Bovine IFN 26~36 15/36~ 6 Numerator = no. alive; denominator = no. treated.

~Cats given human alpha-IFN had significantly ~P<.05) higher survival rates at 6 and 12 months after treatment than cats given bovine IFN. Significance was dPtermined by Chi Square test.

Percent survival of ~linically ill cats positive for FeLVO
Months Af~r Trçatment_ ~ _ Trea~men~ . 6 12 Human alpha-IFN 76% 66~ 61%~
Bovine IYN 72~ 42% 36%
His~rical Control <50% _ <~0% --Numerator = no. aliYe; denominator = no. treated.

~Cats gi~en human alpha-~FN had significantly (P<.05) 1 320qO5 -2~-higher surviYal rates at 6 and 12 mon~hs after tr~atment than cats given bovine IFN~ Signifi~ance was ~etermined by Chi Square test.

TAB~ 3 Response of CPV di~ease to treatment with bovine interferon or human alpha-interferon, by veterinarian.

Attending B~vine I~N Beta ~uman Al~ha XFN
Veterinarian Lived ~ied Lived Died S 16~21 5f21 14/16 2/1 15R 7~1Q ~/10 3/~ __ 0/3 29/42(6~ 42~ 2_ 2~26(~2~ 2/26(~~

Dogs treated with human alpha-interferon had a significantly (P<.05) higher survival rate compared to dogs treated with bovine IFNo Significance between groups was determined by Chi Square test.

Treatment days for CPY disease.
No. o Av~raqe ~o. Treatment Survival Trea~ment _Pays __Days~ SD*~ Rat~
Bovine IFN42 3.31 1.~5 69%
H~ma~ alpha IFN 26 2.75 _ O.92 92%

`. ~

*Calculated on surviv;~g dogs only.
~*Stand~rd deviat~on of the mean treatment d~ys.

Canin~ ~erpqsvixu~ Ch~llen~e Q~ ~ewb~rn ~

Canine herpes~irus infection o dog~ le~s than one week of age are invariablY fatal, but older pups usually survive. Interferon has been successfully used to treat viral inf~ctivns of many species. These studies were conducted to assess the efficacy of interferon in canine herpesvirus (CHV~ ;noculated puppies.
Five (5) pregnant bitches were obtained from a lS USDA licensed supplier and were housed ;n a USDA
approved research facility in Canyon, Te~as. After the pllpS whelped, they wer~ inoculated with 6.3 log 10 units of virulent CHV obtained from Dr. Richard Mock of the Te~as A&~ University Veterinary Medical Diaynostic Laboratory (TVMDL) in Amarillo, Te~as. Human alpha-int~rferon (HAI) or placebo was given to pups orally as treatment in an effort to increase t~e survival rat~ of the CHV inoculated pups. Each litter was divided into control and treated animals. The procedures and schedule for each litter are discussed below. All dead animals were necropsied at TVMDL, Amarillo, Te~as.

LITTER 1:
Nine (9) pups were inoculated orally with CHV
on the day of birth. Interferon was given at 6-10 u~its 1 3~09()5 (lX), or ten ti~es the dosage at 60-100 units (lOX).
~hree pups were given 0.5 ml placebo, 3 pups were given 0.5 ml ~AI (lX), and 3 pup~ were given 0.5 ml of a lOX
co~centrate of HAI orally ~wice daily for 7 days (if they lived that lon~). The 3 controls died 5, 6, and 8 dayæ after CHV inocu~ation. The 3 pups given HAI (lX) l;~ed 7, 7, and 9 dayc and the 3 pups yiven lOX ~AI
li~ed 6, 7, and 7 ~ay~ after CHV inoculation.
Pups given ~AI ~lX) live~ an average of 1.3 days longer than controls, but the longer survival time was not ~tatistically significantO The higher dosage, HAI (lOX), did not provide a survival benefit over the lower dosage, but instead pups given the higher dosage died, on the average, o~e day sooner.
LITTER 2:
Eight (8) pups were inoculated with CHV orally 2 days after birth. Interferon was giYen at 6-10 units (lX), or ten times the dosage (lOX), or l/lOth the dosage (l/lOX). All interferon was given orally after dilution in PBS. Two (2) pups were given 0.5 ~1 PBS, 2 were given O.5 ml HAI at l/lOth concentration, 2 were given HAI (lX~, and 2 were given a lOX concentrate of HAI. All treatments were giYen orally twice daily for S
days starting 1 day beore CHV inoculation~ The 2 controls died <1 and 9 days after CHV inoculation and the HAI treated ~l~lOth dose, full dose, lOX dose) pups died 8 and 9, 5 and 9, 8 and 8 day~ after CXV
inorulation, respectively.

~ o bene~it from treatment at any do~age was ~een. The death of a co~trol pup within a day after CHV
i~oc~lation was probably not relat~d to CHV inoculation.

LITTER 3: .
Nine (9) pups were inoculated with CHV orally 3 - days ater birth. Two pup~ were given 0.5 ml PBS, 2 were given 0.5 ml HAI ~lX), 2 were given l/lOth dose HAI, and 3 were given 2 IU of recombinant human alpha-intereron from Schering-~lough. All treatments were given orally twice daily for 5 days starting two days before CHV inoculation. Both pups given HAI (lX) survived until necropsied 19 days after CHV
i~oculation. One control pup died 14 days after CHV
inoculation and 1 survived until necropsied 19 days after CHV inoculation. Pups given l/lOth dosage of HAI
died 8 and 13 days aft~r CHV inoculation. Only one pup qiven recombinant human alpha-interferon died ~12 days after CHV); the other 2 pUp5 survived until necropsied 19 days after CHV inoculation.
These pups, inoculated 3 days after birth, did not develop an overwhelming CHV infection (only 1 of 2 controls died). A l/lOth dose of HAI did not protect either pup but both XAI ~lX) tr~ated pups survived.
~ITTER 4:
Fourteen (14~ pups were inoculated orally with CHV 2 days after birth. Seven ~7) pups were given PBS
and 7 pups were given HAI (lX) orally twice daily for 7 day~ Sif they lived that long~ starting 2 days after CHV

' ~ ' 1 320~05 inoculation.. The 7 ~ontrol~ ~ie~ 1, 5, 7, 8, 8, 9, and 9 days after CRV inoculation. One of the ~AI (lX3 treated pups survived and the other ~ pups died 1, 6, 8, 9, 9, and 12 days after CHV inoculation~ The deaths of 2 pups only 1 day after CHV inoculation were probably no~ related to CHV inoculation.
One HAI (lX) treated pup lived 3 days beyond the last surviving control and one HAI (lX3 treated pup lived 2 weeks (until necropsied) beyond any treated control pup. Average survival time of intexfcron treated pups was longer than control survival time, but not signif icantly so.

LITTER 5:
Si~ ~6) pUp5 were inoculated orally with CHV 2 days after bîrth. Three ~3) pups were given PBS and 3 were given HAI (lX) orally once daily starting 5 days after CHV ino~ulation. The 3 controls died 6, 6, and 7 days after CH~ inoculation. One of the HAI (lX) treated pups survived (until necropsied) and the others died 8 and 9 days after CHV inoculation.
All interferon treated pups lived longer than any of the control pups. Treatment with HAI (lX) did not begin until 5 days after C~V inoculation, yet survival was significantly (Pc.05) prolonqed.
In summary, on the average, puppies treated with human alpha-interferon had longer survival times and enhanced survival rates compared to littermate controls, after canine herpesvirus challenge. A total of 7 puppies (1 control and 6 interferon treated) 1 ;~20905 ~urviv~d the normally ~atal CHV inoculation. The data is ~ummarized in the Table S below.

T~LE 5 Summary of Canine Herpesvirus Data ~verage No. of Survival ~i~ter PUpa Dosa~e. ime (Day~ yjy~
1 3 control 6.33 0 1 3 HAI lX 7.67 0 1 3 HAI lOX- 6.67 0 2 2 control 4.5** 0 2 2 HAI l~lOth 8.5 0 2 2 HAI lX 7.0 0 2 2 HAI lOX 8.0 O
3 2 control 14.0 3 2 HAI l/lOth 10.5 0 3 2 HAI lX - 2 3 3 recombinant IFN 12.0 2 4 7 control 6.7 0 4 7 HAI lX 7.5 3 control 6.3 0 3 HAI lX 8.5 * dead dogs survival time; living puppies ~ot calculated.
~* includes one pup living only one day beyond CHV
inoculation.

, ~ ; .

.
- . .
, - - -1 320~05 T~eatm~a~l~f ~a~ lar. ~erm~

Three ~ases of nasal solar dermatitis (collie nosej clea~ed after human alpha interferon treatment of 1 uni V lb body weight orally and topical treatment (a few ml at 20 units/ml).

~rea~m~n~_~ Ganinç ~pus ~ryt~ tQsus Two cases diagnosed as canine lupus erythematosus were cured by human alpha-interferon treatmentO A 2 year-old Lhasa apso male had been treated with prednisolone for 1 year for 3 dermatological lesions on the abdomen and prepuce. The flat glisten;ng lesions were continually licked by the dog. Within 1 week of oral human alpha-interferon treatment ~1 uni V lb body weight daily for 5 days, then after 1 week, treatment was repeated for 5 days) 2 lesions completely healed and the third lesion was reduced to 1/2 its original size. Within 4 weeks, the lesions were all completely healed and all th~rapy ceased. One year later, a skin lesion reappeared but promptly healed after interferon treatment was repeated. The skin l~sions have not reappeared in the past 10 months.
A 6 year-old spayed female chihuahua cross had a spider shaped ~4 cm by 2 cm appro~imately) skin lesion on the abdomen. The lesion was flat, glistening and pruritic. Si~ weeks of prednisolone treatment resulted in complete healing. The following summer, the lesion .' '' ' ' .
.

' 'I 320qO5 ~27--reappeare~ an~ was treated with oral human alpha-inter~eron at about 1 urii~,/lb ~ody weight daily for S d~y~. W;thin 5 days th~ lesion was reduced to 1/3 it~ original ~ e and completely disappear~d within 10 days. The lesion has not reappeared in the past year.
Treatmenlt O Feline Inf~ç~ s Peri~nit:is Table 6 shows th~ result~ of 17 case~ of feline infections p~ritonitis (FIP~ as diagnosed by practicinq veterinarians. Human alpha-interferon ~IFN~ treatment resulted in a significantly greater survival rate than ~reatment with bovine beta-IFN.

TABLE ~
Survival of clinically ill cats diagnosed as FIP
No~ Cats SurYival Tre.~ment _ Trea~ed _ _ AliYeDead Rate Human alpha-IFN 11 10 1 ,91%
Bovine beta-IFN 6 3 3 50%
TQ~al _ _17 1~ _ 4 . _76% _ Cats given human alpha-IFN had a significantly (P=.0574) greater survival rate than ~ats given bovine beta-IFN.

Human Trç~tme~ Wi~h ~Qaenous Human pat;ents wer~ treated with human alpha interferon in the therapy of acute rheumatoid arthriti~, multiple ~clerosi~, a ~hma, acne~ malignant lymphoma, mesothelioma, an~ apthous ~tomatit;s. Therapy consisted of oral a~ministration of 0.7 IU pPr lb. of patient body weight t~ice daily, once in the morning and once in the evening. None of the patients noted any fever or anore~ia associated with the administration of alpha intçsferon. Interferon was administered in a buffered solution having a concentration ~uch that a single dosage could be administered in a volume of about 1 to about 20 ml of liquid. Each patient generally retained the interf~ron solution in his mouth for a per;od of time up to about one minute. After that time the solution wa~ either ~wallowed or discharged from the patient's mouth.
Two patients suffering from rheumatoid ~rthritis were treated -- a Caucasian male age 44 and a Caucasian female age ~4. The male patient was pain free in 7 days, and the female was pain free in 10 days.
They were both continued on the oral intereron for 21 days total and have remained asymptomatic.
It has been found that recurrence of a tr~ated arthritic condition can be minimized if treatment in accordance with the present invention is continued over a period of up to about three months.
A ~0-year-old Caucasian female nurse afflicted with multiple sclerosïs and who had had an e~tensive , '~

neurologi~ workup at City of ~ope ~o~pital in Los ~ngele~ recei~ed treatme~t in accordance with the pre~ent invention for 21 ~ay~. The patient has had no recurrence of h~r n~urologic symptoms for the past nine months.
A 42-year-old Caucasian male diagnosea ~o have a malignan~ lymphoma had completed chemotherapy with dismal results and was considered terminal. He was treated for three weeks with oral in~erferon. ~i~
months after starting treatment he was r~leased by his oncologist as free of the disease.
An 82-year-old Caucasian female was diaynosed to have mesothelioma~ Presently there is no effecti~e treatment for that disease ~nd only a 9-month average survival rate is predicted. During her trPatment with human alpha-interferon ~he ha~ thoracente~is on two occasions for plural effusion, Otherwise, the patient has been actiYe and has survived for 43 months.
A 32-year-old Asian male with apthous stomatitis was treated for two weeks with human alpha-interferon in accordance with the present invention. There has been no recurrence of the ulcers over the last si~ months since treatment was completed.
BKC is a 29 year-old Caucasian female and KKJ
is a 20 year old Caucasian emale. Both are afflicted by acne-like ~kin blemishes at the time of their monthly menstrual cycle. Oral human alpha-interferon given at about 1 unit/lb of body weight for 3 days prior to the time of their cycle reduces the ~everity and number of skin blemishesO

1 320~05 -3~-T~a~me~t~ ma~
Wi~h ~ovln~ h~h~-ln~er~erQn S MA~, a 38 year-old C~ucasian Caucasian female, had 7 war~ on ~he middl~ finger of her right hand.
A~ter 9 months duration, medical treat~ent was ~ough~, and liquid nitrogen was applie~ by a ~ermatolog1st.
Only one wart on the finger regressed after treatment.
Three warts coalesced to create a large wart area tha~, over the ne2t year, acquired a roughly 12 millimeter square shape. Oral bovine alpha interferon treatment wa~ started at a dosage of 6 ml daily for 6 consecutive days. The concentration of alpha-interferon was 30 units/ml; it was derived from the nasal secretions of cattle infected with infectious bovine rhinotracheitis virus. All the warts completely regressed within 6 weeks of the first dose of interferon.

Tn~f~ron DQsag Q Form~ ions (1) Lozenge A starch gel-based lozenge containing interferon is prepared by combini~g 150 grams of ~ucrose, 550 ml phosphate buffered saline, and 250 grams o a cold-water-soluble starch such as that described in U.S~ Patent 4,465,702, heating that misture with stirring to a temperaturs of ~bout 75C, cooling the mi~ture to about 30C and thereafter blendang into the 3~ paste-like mass S0 ml of phosphate buffered sal;ne PBS

. ~ - .

~, 1 ~0905 containing human alpha interferon at a concentration of 250 IU~ml. The mi~ture is th~n formed into multiple portions of about 5 to about 10 grams each which set upon ~tanding under drying conditions to a starch candy gel-like consiste~cy. The lozenges therehy produced can be administered to a patient singly or in combination.
The patient is in~tructed to hold the lozenge in his mouth until it is completely dissolved to release the interferon component for contact with the oral mucosa.

(2) Chewable Yitamin A chewable vitamin formulation is prepared, for e~ample, according to the description of U.S. Patent 3,857,939 by coating one or more components thereof prior to ta~leting with an interferon solution in an amount sufficient to provide about 1 to about 1500 units of interferon in each chewable vitamin tablet.

(33 Mouthwash A mouthwash formulation is prepared in accordance with the pre~ent invention by combiPing 850 ml PBS, 100 ml o glycerin, 50 grams of de~trose, and a misture of 0.3 ml of a flavor oil pre-mi~ed with 30 ml of a palatable, pharmaceutically ~cceptable surfactant/dispersant having an HLB from about 15 to about 25 and 50 ml of a PBS solution of interferon (concentration 120 IU/ml). The formulation contains interferon at a concentration of about 120 IU per 20 ml dosage. The patient is asked to hold a 20 ml volume of the mouthwash in his mouth, optionally gargling with the same, for a period of about 15 seconds to about one minute.

(4) Syrup Interferon is added to a commercial cough syrup formulation in an amount ~ufficient to provide an interferon containing syrup formulation having about 1 to about 1500 IU of human interferon per tablespoon of SyrUp.

(53 Effer~escent Tablet A ta~leting misture comprising a pharmaceutically acceptable alkali metal carbonate o~
bicarbonate, an organic acid such as citric acid, human interferon (preferably dispersed on a suitable organic or inorganic carrier therefor) in an amount sufficient to provide a per tablet dose of about 1 to about 1500 units of intereron per dose, and further i~cluding suitable ~ableting ~cipients such as lubricants and binders, is compressed into a unitary dosage fQrm of interferon. The compressed tablet efervesces upon contact with water to release interferon to the resulting buffered solution. The dosage of interferon is readily available in ~olution for contact with the oral pharyngeal mucosa of a patient in need of said dosage of interferonO

Claims (16)

1. What is claimed is:
   l. An immuno-therapeutic oral dosage form of interferon for human use, said dosage form consisting essentially of interferon in an amount corresponding to about 0.01 to about 5 IU of interferon per pound of body weight, and pharmaceutically acceptable excipients selected to promote contact of said dosage of interferon with the oral and pharyngeal mucosa of a patient receiving said dosage form to stimulate a systemic immuno-therapeutic response.
2. 2. The dosage form of Claim 1 in the form of a mouthwash.
3. 3. The interferon dosage form of Claim 1 in the form of a lozenge specified for dissolution during con-tact with the saliva in the mouth.
4. 4. The lozenge of Claim 3 formed as a compressed tablet containing about 1 to about 1500 IU of human alpha-interferon or human beta-interferon.
5. 5. A chewable tablet in accordance with Claim 3.
6. 6. An immuno-therapeutic oral dosage formulation of interferon in the form of an effervescent tablet for use by a human patient, said dosage form being formulated to release upon effervescent dissolution in water about 0.01 to about 5 IU of interferon/lb of patient body weight.
7. 7. The interferon dosage form of Claim 6 wherein the interferon is human alpha-interferon and said dosage form is formulated to provide about 10 to 1500 IU of alpha-interferon per dose.
8. 8. The interferon dosage form of Claim 1 in the form of a syrup containing about 1 to about 1500 IU of human interferon per tablespoon.
9. 9. The immuno-therapeutic dosage form of Claim 1 wherein the interferon is human alpha-interferon and said dosage form is formulated to provide upon administration about 10 to about 1500 IU of alpha-interferon for contact with the oral and pharyngeal mucosa.
10. 10. In combination, for treatment of humans afflicted with immuno-resistant disease characterized by apparent hyperactive or hypoactive immune system function, an immuno-therapeutic dosage form of interferon for use by a human patient, said dosage form comprising about 0.01 to about 5 IU of interferon per pound of patient body weight and a pharmaceutically acceptable excipient allowing contact of the interferon with the oral and pharyngeal mucosa of said patient upon per os administration of said dosage form, and instructions to said patient to hold said dosage form in the mouth to promote contact of the interferon with the patient's oral and pharyngeal mucosa.
11. 11. The combination of Claim 10 wherein the dosage form contains about 1 to about 1500 IU of human alpha-interferon or .
    human beta-interferon.
12. 12. The combination of Claim 10 wherein the interferon is alpha-interferon.
13. 13. The combination of Claim 10 wherein the interferon is human leukocyte interferon.
14. 14. The combination of Claim 10 wherein the interferon is beta-interferon.
15. 15. The combination of Claim 10 wherein the amount of interferon in the dosage form is about 0.1 to about 4 I.O. per pound of patient body weight.
16. 16. The combination of Claim 10 wherein the dosage form is a solid dosage form adapted for dissolution during contact with saliva in the mouth.