SE9701212L - Use of bird antibodies - Google Patents

Abstract

Use of avian antibodies is claimed for production of a drug for treatment and/or prevention of respiratory tract infections.

Description

1 USE OF AVIAN ANTIBODIESink. t. Patent- och regmerket 1997 -04- 03 Technical fieldHwudfaxen Kassaa The present invention relates to use of avian antibodies against infectious antigen, and/or antigen binding fragments thereof, for the production of a drug for local treatment and/or prevention of infection in individuals having increased susceptibility to infections.

Background of the invention and prior art Chronic colonization with 12"sipudom9nas aeruginosa in the respiratory tract of patients with cystic fibrosis (CF) is a principal cause of the high morbidity and mortality in this disease. It is very difficult to get rid of Pseudomonas aeruoinosa once it has been isolated from the sputum of CF-patients. Only a temporary eradication of this pathogen can be achieved by vigorous antibiotic treatment during very early phases of colonization - and the bacteria will return very soon (1-3).

The best results hitherto have been reported from the CF-center in Copenhagen (2) where 14 CF-patients were treated daily with oral ciprofioxacin and inhalation of colistin for 3 weeks at their first Pseudomonas aeruainosa-positive culture. After treatment, these CF-patients were observed for up to 27 months (totally 214 months; mean 15.3 months). During this time 2 of the patients became chronically colonized with Pseudorportes sermsinpse (defined as six consecutive sputum cultures positive for the bacteria) and there were 49 sputum cultures positive for pseudomonas aeruoinosa out of 214 (=23%).

From another Copenhagen study it is reported that Pseudomonas aeruginosa reoccured in sputum cultures already 4 months after a course of anti-pseudomonas chemotherapy in 98.3% of the CF-patients (3) Therapeutic and prophylactic use of antibodies from eggs of hens has several important advantages. Eggs is a common foodstuff, eaten almost daily by many eli—Ht.-1( 14.

*CHM rHurroiLH40 10D007...) 011.0 04 Ink. t. Patent- och reg.verket 1997 -04-03 )iirmdkona Kass=2 people. Thus, antibodies from eggs could be used without any side effects by all people except those few who are allergic to them. Eggs contain very high concentrations of antibodies and they are easy to obtain. Hens, immunized with specific antigens, produce eggs with high antibody titers against these antigens (4). The production of antibodies in eggs from one hen during one week is equal to the amount produced in 180 cc of blood from mammals.

In contrast to antibodies from mammals (eg bovine), the antibodies from eggs (5) do not activate the human complement system (6). This is a tremendous advantage since activated complement factors are very effective mediators of inflammatory reactions. In addition, antibodies from eggs do not react with rheumafactors (7), human Fc-receptors (8), bacterial Fc-receptors (9) or human anti-mouse IgGantibodies (10), which make them very safe to use.

In several trials, antibodies from eggs have been tried to prevent or treat bacterial or viral infections in the digestive tract of different animals with promising results (1115).

However, no such results have been shown on humans with infections caused by infectious microbes. Hitherto, individuals having increased susceptibility to infection have had to rely on conventional therapy, such as antibiotic treatment. Furthermore, this conventional treatment is often systemically administred causing undesired side effects.

Summary of the invention The present invention suggests, for the first time, use of avian antibodies to locally prevent and treat infections in humans.

The present invention also suggests, for the first time, that use of avian antibodies can prevent infections in other sites than administred, eg local peroral treatment prevents lung infection.

-.HrKi,177( 14.4biCANN M4IJ1I urriri4m41D 10dSJS SW IdOtio Ink. 1. Patent- och reg.verket 1997 -04- 03 livvirdfaxen Kassan3 Thus, the present invention relates to use of avian antibodies against infectious antigen, and/or antigen binding fragments thereof, for the production of a drug for local treatment and/or prevention of infection in individuals having increased susceptibility to infections.

In one embodiment of the invention, the infection is a respiratory tract infection, for example a lung infection, caused by Pseudomonads and/or related bacteria, such as Pseudomonas aeruginosa in an individual with cystic fibrosis.

In this embodiment, the local treatment or prevention is preferably at the lymphatic ring in oropharynx by gargling, swallowing or by inhalation.

In a second embodiment of the invention, the infection is a skin infection caused by Pseudomontsjs and/or related bacteria. Such skin Infections are common in individuals with thermal injury. In this embodiment the avian antibodies are locally adminstred to the site of the injury in a suitable vehicle, such as an ointment or cream etc.

In a third embodiment, the infection is a fungal Infection caused by Canclida and/or related microbes in an individual having increased susceptibility to infection due to antibiotic and/or cytostatic treatment . The fungal infection is often localized to the mouth and/or vagina. In this embodiment the avian antibodies are locally administred to the mouth or vagina in siutable vehicles. For vulva vaginitis applications, vaginal inserts is a suitable administration form.

Detailed description of the invention At their first colonization with Fleudomonas aerueinosa in the respiratory tract, two CF-patients were treated with the oral antibiotic ciprofloxacin and antibiotic inhalations of colistin or tobramycin for three weeks. Simultaneously with the antibiotic treatment, they started to gargle daily (for 2 minutes) and thereafter to swallow specific antibodies against Pseudomonas aeruninosa. After this three week 0.1-HrX.-1V.Vf 14.4f1.1.KMNNurriqLm40. 1=007J, WU 00 :•.• •••• lnk L Patent. och reg.verlet 1997 -04-03 Huvwfaxan Kos=4 period, the patients have continously received antibodies according to the above procedure, but without antibiotics, for together more than 30 months (26 and 4 months, respectively). All sputum cultures during the treatment period have been negative for PseudorponaEteruoinosa. (0 positive out of 28 0%; this should be compared with the results from the Copenhagen study, referred to above, which was 49 out of 214 = 23% p < 0.005). Thus, a complete eradication of Pseudomonas apruoinosa in the respiratory tract of CF-patients is achieved. The results strongly suggest that the treatment with gargling and swallowing of antibodies against Psiudornonas aeru2inosa according to the invention is effective to prevent chronic colonization of the bacteria. The effect is with all probability due to a local effect of the avian antibodies against bacteria in the lymphatic ring in oropharaynx which capture' the bacteria and destroy them.

Materials and methods Each egg from a hen contains more than 100 mg antibodies. These antibodies are produced by hens and transported to their eggs where they are found in high concentrations. High titres of specific antibodies against bacteria were achieved by repeated immunization of hens with killed specific bacteria or fragments thereof. Specific antibodies against Pseudorriquas geruginosa have been produced by repeated immunizations of hens with killed Ps - • • u -nes aeruainosa. Eggs from these hens have been used to make a solution with high specific antibody concentration.

For CF-patients, the dose is preferably about 50 mg IgY per day. Rigorous precautions have been made to avoid bacterial contaminations of the solutions, which thereafter have been kept at -20° C. The CF-patients have been asked to take Out a bottle with 30 cc of the solution from the freezer each morning and to gargle with this solution in the evening for 2 minutes and thereafter to swallow it.

Case reports • a • • a • a • 11.5-Hritr1Y 14;4(eWItNI urri.4.14401.13.1.1) UY Ink. I. Patent och reg.verld 1997 -04- 0 3 Huvudfcccen Kauai Case 1. A 21 year old female with CF. Diagnosed at birth by screening with albumin in meconium (179 mg albumin/ g dry weight meconium) and subsequent sweat test (Na 101, Cl 122 mmolfkg sweat). Already during her first three weeks of life there were severe feeding problems. She had frequent stools and was vomiting considerably and her weight went down from 3430 g to 2930 g despite a vigorous appetite. After proper therapy (pancreatic enzymes, breast feeding, extra vitamins, mist tent, physical therapy and anti-staphyloccal antibiotics at every respiratory infection) was instituted she started to gain weight and was doing quite well. Her height was steadily slightly above the mean for her age to a final height of 169 cm; her weight was usually at the mean for her age but had a severe dip at 7-8 years of age and she did not come back to mean weight until 15 years of age. During the last two years her weight has been around 53 kg, ie 8M1 (body mass index) 18,6. From 7% years of age she has always had ZtgitxisraagsmaiL_Ireus in her sputum and occasionally also Hemophilus infiuenzaa or Proteus mirabilis. Her chest X-rays showed a slight but steady progress of typical CF-changes and her lung function deteriorated slowly to FVC (forced vital capacity) of about 75%, and FEV1 (forced expiratory volume in one second) to about 50% of predicted values at the age of 19 years. Her first colonization with Esludomonas aeruginosa occurred in November 1989. This was effectively treated with azactam and gentamycin iv in two periods of fourteen days each and addition of ciprofloxain orally at the second course. Thereafter her sputum cultures returned to the earlier pattern of Staohvloccus aureus and liemoohilus infiuenzae until August 1994. At this time the bacteria were eliminated after a 14 days course of ceftazidim and tobramycin iv.

However, already in January 1995, Pseudomonis agruoinosa was again found in her sputum. At this time it was decided to give her an antibiotic course according to the Copenhagen model (2) with ciprofloxin 750 mg x 3 orally and colistin 2 million U x 2 by inhalation for three weeks and at the same time start with daily gargling and svrallowIng of avian antibodies against Pseudomonas aeruoinosa. The daily intake of antibodies has continued since then (now for 26 months). There has been no new appearence of Pseudomonas aeruoinose and the pattern of bacteria in her sputum • • • %• .1IJA.Nrs.r. lort .6.11um- r.14 .1.1.A.11‘14•••4 .14 1 .lo• R.114 :• •••• • • • • • Ink t, Patent- och reg.vsket 1997 -04- 0 3 Huvudfccon Kama6 has returned to the usual. She has never had any precipitins or antibodies against Psedomonas aeruoinosa in her serum. No side effects of the treatment have been seen in her blood (red and white blood cells, trombocytes, liver enzymes or creatinine values). Her chest X-rays have been essentially unchanged since 1995 as well as her lung function tests (FVC still 75% and FEV1 now 45% of predicted). She is an extremely active woman and her working capacity (150 Watt) must be considered very good in regard to her severe disease.

Case 2. A 17 year old girt with CF. During her first two years of life she had recurrent obstructive bronchitis, frequent greasy, foul smelling, loose stools and poor weight gain (weight 9,5 kg at 2 years of age). At this time, sweat tests were performed and reveled the diagnosis of CF. Intensive treatment was then instituted. Her weight came back to mean values but her height followed the -2 SD curve and has stopped at 151 cm. Her main problem has been recurrent stomach ache of the type "distal intestinal obstruction syndrome (DIOS)". Her lungs have always been very good - chest X-rays show only minimal changes, lung function tests have all been at or above mean for her age and height (the exception is RV (residual volume): 1,14 = 186% of predicted). Her working capacity (120 Watt) is nearly normal. Her sputum cultures had always shown Staphylococcus aureys and/or Hemophilus influenzae until November 1996 at which time the cultures for the first time showed pseudo'rnonas aerurainose. She then immediately got a three weeks course of ciprofloxacin 750 mg x 2 orally and tobramycin 320 mg x 2 by inhalation (colistin was not available). Simultaneously she started daily gargling and swallowing of avian antibodies against Pseudomonas aeruginosa with which she has continued since then. Pseudomonas was eradicated and since then (now for 4 months) she has had her usual bacteria in her sputum. She continues to do well.

In summary, even if only two patients were included in the study, the results strongly suggest a prophylactic and therapeutic effect of the treatment with avian antibodies against Pseudomonas aeruoinosa according to the invention. In 30 sputum cultures during the therapeutic time period of the two patients described above no DIAMNN rmitml ut1r.4m6mso ltdobtov,av UJ Ink. I. Patent• nth reg.vetlet 1997 -04- 03 Huvudfaxan Kassa7 Pseudomortgs aeruabusq positive cultures have been found. In contrast, in the study from Copenhagen referred to above, there were 23% positive cultures, and as many as 75 - 100% of these patients had at least one positive culture during a similar observation period as in the present invention. 03-APR-1997 14;49BRAN PATENT UPPSALA46 1U568SJ9 SID 116 Inks t Pate* och reg.verket 1997 —04— 0 38 REFERENCESkluvudfaxen Kaman 1.Haiby N., Pseudomonas infection in Cystic fibrosis, In Cystc Fibrosis, Current topics: Vol. 1, edited by Dodge IX, Brock D.J.H. & Widdicombe J.H., John Wiley & Sons Ltd., London, 1993, pp 251-268. 2_Valerius N.H., Koch C., Heiby N., Prevention of chronic Pseudomonas aeruginosa colonisation in cystic fibrosis by early treatment The Lancet 1991; 338; 725-726 3.Szaff M., Heiby N. and Fiensborg LW.; Frequent antibiotic therapy improves survival of cystic fibrosis patients with chronic Pseudomonas aeruginosa infection; Acta Peediatr Scand 72:651.657,1983. 4,Larsson A., BalOw R-M., Undahl T.L. and Forsberg P.-O. (1993) Chicken IgG: Utilizing the evolutionary advantage; Poultry Science, 72, 1807-1812.

Larsson A. and Lindahl T.L (1993) Chicken antibodies: A tool to avoid interference in immunological assays; Avian immunology in progress, 62,97-102.

Larsson A., Wejiker P.-E., Forsberg P.O. and Lindahl T.L. (1992) Chicken antibodies: A tool to avoid interference by complement activation in ELISA; J. lmmunal. Methods (1992) 156, 79-83.

Larsson A. and Sj6quist J. (1988) Chicken antibodies: A tool to avoid false positive results by rheumatoid factor in latex fixation tests; J. Immunol. Methods 108, 205-208.

Lindahl IL, Festin R and Larsson A (1992), Studies of fibrinogen binding to platelets by flow cytometry: An improved method for detection of platelet activation; Thrombosis and Haemostasis, 68,221-225. 101,Mr1.0-1VVr 14.447MMOINN rpIIrI urrQmLm40 100000J7 011/ 11 hicL patent- oth wallet 1997 -04- 0 39 ihroxffinan Kamm Larsson A. and Lindahl T.L, (1993), Chicken anti-protein G for the detection of small amounts of protein G.;Hybridoma, 12,143-147.

Larsson A. and Mellstedt H. (1992), Chicken antibodies: a tool to avoid interference by human anti-mouse antibodies in ELISA after in vivo treatment with murine monoclonal antibodies; Hybridoma 11, 33-39. 11,Bartz C.R., Conklin R.H., Tunstall C.B. and Steele J.H., Prevention of murine rotavirus infection with chicken egg yolk immunoglobulins; J. Infect. Dis., 142: (1980) 439.

Ebina T., Tsukada K ,Urnezu K, Nose M., Tsuda K, Hatta H., Kim M. and Yamamoto T., Gastroenteritis in suckling mice caused by human rotavirus can be prevented with egg yolk immunoglobulin (IgY) and treated with a protein-bound polysaccharide preparation (PSK); Microbial. Immunal., 34: (1990) 617.

Hiraga C., Kodama Y., Sugiyama T. and Ichikawa Y., Prevention of human rotavirus infection with chicken egg yolk imrnunoglobulins containing rotavirus antibody in cat; J. Jpn. Assoc. Infect. Dis., 156: (1990) 118.

Ikemori Y., Kuroki M., Peralta R.C., Yokoyama H. and Kodama Y., Protection of neonatal calves against fatal enteric oolibacillosis by administration of egg yolk powder from hens immunized with K99-piliated enterotoxigenic Escherichia coli; Am. J. Vet. Res., 53: (1992) 2005.

O'Farrelly C., Branton D. and Wanke CA., Oral ingestion of egg yolk immunoglobulin from hens immunized with an enterotoxigenic Escherichia coil strain prevents diarrhoea in rabbits challenged with the same strain; Infect. lmmun., 60: (1992) 2593. 14,4,WC•KM111•4 rMIMMI wmr=ma-ri.448. 1000e7J7 QAW 14 InktPatentochreg.verket12 1997 -04- 0 3 Nvvudkown KammABSTRACT The present invention relates to use of avian antibodies against infectious antigen, and/or antigen binding fragments thereof, for the production of a drug for local treatment and/or prevention of infection in individuals having increased susceptibility to infections. An example of such indivudals is individuals with cystic fibrosis who often have severe respiratory tract infections caused by Pseudomonas aeruginosa.

ANT.SID 14

Claims (15)

CLAIMS 1. Use of avian antibodies against infectious antigen, and/or antigen binding fragments thereof, for the production of a drug for local treatment and/or prevention of infection in individuals having increased susceptibility to infections. 2. Use according to claim 1, wherein the infection is a respiratory tract infection. 3. Use according to claim 2, wherein the infection is a lung infection. 4. Use according to claim 2 or 3, wherein the infection is caused by Pseudomonads and/or related bacteria. 5. Use according to claim 4, wherein the infection is caused by Pseudomoges a91-01129, 6. Use according to any one of the claims 1-5, wherein the individual having increased susceptibility to infection is an individual with cystic fibrosis. 7. Use according to any one of the claims 1-6, wherein the local administration for treatment or prevention is at the lymphatic ring in oropharynx 8. Use according to claim 7, wherein the local treatment or prevention is performed 1. •• •by gargling, swallowing or by inhalation. 2. •• • 9. Use according to claim 1, wherein the infection is a skin infection. 10. Use according to claim 9, wherein the infection is caused by Pseudornonad4 1. • and/or related bacteria. 2. • 3. • • *• 4. • • 5. I• :.• 6. IP tx-r-firmY-Iv'turrapiLm100007,)0 =AV 1.) Ink.t.Patent- och remit 1997 -04- 03 tluvudkran Kowa11 It Use according to claims Sot 10, wherein the individual having increased susceptibility to infection is an indivual with a thermal injury. 12. Use according to claim 1, wherein the infection is a fungal infection. 13. Use according to claim 12, wherein the infection is caused by candiga and/or related microbes. 14. Use according to claims 12 or 13, wherein the individual having increased susceptibility to infection is an indivual under antibiotic and/or cytostatic treatment . 15. Use according to claims 12, 13, or 14, wherein the infection is vaginal or oral 0•

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7. • • 14,4,WC•KM111•4 rMIMMI wmr=ma-ri.44

8. 1000e7J7 QAW 14 InktPatentochreg.verket12 1997 -04- 0 3 Nvvudkown KammABSTRACT The present invention relates to use of avian antibodies against infectious antigen, and/or antigen binding fragments thereof, for the production of a drug for local treatment and/or prevention of infection in individuals having increased susceptibility to infections. An example of such indivudals is individuals with cystic fibrosis who often have severe respiratory tract infections caused by Pseudomonas aeruginosa. ANT.SID 14