Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
  • A01N1/10—Preservation of living parts
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
  • A01N1/10—Preservation of living parts
  • A01N1/12—Chemical aspects of preservation
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
  • A01N1/10—Preservation of living parts
  • A01N1/12—Chemical aspects of preservation
  • A01N1/122—Preservation or perfusion media
  • A01N1/124—Disinfecting agents, e.g. antimicrobials
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/56—Materials from animals other than mammals
  • A61K35/62—Leeches; Worms, e.g. cestodes, tapeworms, nematodes, roundworms, earth worms, ascarids, filarias, hookworms, trichinella or taenia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • 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
• • 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
  • A61P37/02—Immunomodulators
• • 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
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants

Definitions

• the present invention relates to a purification process for the preparation of viable, viable suspensions containing eggs of parasitic helminths and suitable for therapeutic use.
• Th1-dominated autoimmune disease can be prevented or compromised by helminth infection (Summers et al., Am J Gastroenterol 2003, 98: 2034-2041).
• Other diseases caused by Th1 immune cells, as well as Helicobacter-induced gastritis and autoimmune encephalomyelitis can be influenced.
• T ⁇ churis suis is closely related to Trichuris humanis and survives in the human gastrointestinal tract without, however, multiplying. Therapeutic measures are not necessary during this self-limited infection. Infection by Trichuris suis can therefore cause immunization.
• RJS Beer (Parasitoiogy 1972, 65: 343-350) describes a method for collecting and cleaning worm eggs. Worm eggs in the embryonated stage are cultured in a 0.2% potassium dichromate solution at 32 ° C and aerated daily. DE 101 63 115 describes a similar cleaning method. There are worm eggs with the help of hydroxyl or oxygen radicals producing chemical reactions freed of any microorganisms and viruses present. Especially the so-called Fenton reaction is described. In this case, under the influence of FeCl 2 from H 2 O 2, oxygen is generated in statu nascendi, whose disinfecting effect is utilized.
• An object of the present invention is to produce an embryonated worm egg suspension which is purified from contaminating viruses and microorganisms, in particular bacteria, and which can be stored without growth of fungal spores and yeasts.
• the methods described here preserve the ability of worm eggs to develop into adult worms.
• the worm egg suspensions are suitable for therapeutic use.
• isolated eggs of helminth preferably of T. suis
• an acid solution preferably an H 2 SO 4 solution
• the optimal incubation time is 2 to 8 hours, preferably 3 hours.
• the incubation is carried out at a temperature of 4 ° C to 37 ° C, preferably 25 ° C to 35 ° C.
• the acid treatment is effected by adding sulfuric acid.
• a sufficient amount of acid is added to lower the pH to a range between about 0.5 and 2.
• the pH is lowered to about 0 to ⁇ 1.
• Suitable acids are hydrochloric acid, Nitric acid or preferably sulfuric acid.
• the acid treatment may only be carried out over a relatively short period of time, so that the worm eggs are not damaged too much.
• the pH is lowered to less than 2, more preferably even to pH 0.6 to 0.8.
• the duration of the acid treatment is a few minutes to a few hours, preferably 120 minutes to 360 minutes.
• the still tolerable short-term period includes the process of embryonating the worm eggs (3 months at 22-25 ° C).
• the pH is raised again to a pH> 4 by addition of a suitable base, for example NaOH.
• a suitable base for example NaOH.
• one or more preservatives is added, in principle, all suitable for the preservation of foods or pharmaceuticals substances can be used. Preference is given to using preservatives which are well tolerated by patients suffering from Crohn's disease.
• the preservative is preferably selected from the group consisting of sorbic acid, benzoic acid, salts of these acids, parabenzoic acid esters, p-hydroxybenzoic acid esters, propylene glycol or combinations of these preservatives.
• weaker acidic solutions with the addition of the cited preservatives, preferably sorbic acid and its salts, are also suitable for the rapid inactivation of microorganisms and viruses.
• the use of these solutions has the advantage over the use of strong acids that the worm eggs can be purified in a single medium due to the less acidic pH, embryonated (3 months at 22-25 ° C) stored and applied to the patient , This long-term use does not compromise viability the worm eggs (see example 5).
• This gentle process uses preservative-containing media having a pH of> 1, preferably ⁇ 2, and does not impose any restrictions on the microbiological purity of the worm egg suspensions.
• the resulting preservative-containing suspensions with embryonated worm eggs are suitable for use on the patient as a drink solution.
• further pharmaceutically acceptable additives such as dyes, flavorings or thickeners may be added to these worm egg suspensions.
• the invention therefore provides orally administrable pharmaceutical preparations containing a storable suspension of viable eggs of parasitic, non-human pathogenic helminths, in particular T. suis, wherein a sufficient number of helminths develops after taking the suspension, which leads to a stimulation of regulatory T cells and wherein the suspension comprises less than 1000 colony forming unit (cfu) microorganisms per ml of suspension.
• the orally administrable pharmaceutical preparation contains less than 100 colony-forming units of microorganisms per ml of suspension.
• the administrable pharmaceutical preparation contains less than 10 colony-forming units of microorganisms per ml of suspension. The number of colony-forming units is determined by conventional microbiological methods.
• Microorganisms are understood here to mean bacteria, viruses, fungi, yeasts and protozoa, although microorganisms which may have adverse health effects may not be present.
• the method according to the invention it is possible to provide the suspension of storable eggs of parasitic, non-human pathogenic helminths in a form which are suitable for pharmaceutical use.
• the treatment according to the invention on the one hand contaminating microorganisms, in particular bacteria, but also fungi, viruses and optionally protozoa are killed to such an extent that the preparation is suitable for pharmaceutical use.
• the treatment is so gentle that the worm eggs retain their ability to grow after ingestion in the patient's intestine, so that the desired stimulation of the immune system can take place.
• the preparations according to the invention are particularly suitable for the treatment of various inflammatory bowel diseases, especially chronic inflammatory bowel diseases. In a particularly advantageous manner, the preparations can be used for the treatment of inflammatory bowel disease, which are referred to as Crohn's disease.
• TSO Trich ⁇ ris suis Eisuspension
• phosphate buffer (pH 7) phosphate buffer (pH 7) in a concentration of 2,400 eggs / ml
• DAB German Pharmacopoeia, Section 2.6.12, last updated with the 24th supplementary delivery in 2006
• a germ status of 190,000 cfu / ml was found.
• This suspension was then rebuffered to pH 2 with dilute H 2 SO 4 solution. After 3 h storage at 30 0 C, the bacterial count had been reduced to ⁇ 20 cfu / ml.
• Examples 1 and 2 ac show that after a short time bacteria are effectively killed with a dilute H 2 SO 4 solution pH 2. Yeasts and fungi require either a longer incubation period or more acidic pH levels to be effectively killed.
• the purification steps described are surprisingly also suitable for the preparation of a virus-free T. su / s ice suspension.
• Murine Leukemia Virus 1 Pseudorabies Virus (PRV), Porcine Parvovirus (PPV) and Feline Calicivirus (FCV) in the Preparation of Trichuris suis Ice Suspension (TSO).
• the viruses used are model viruses.
• the virus titer in the inoculated samples was determined by endpoint titration.
• dilute samples are applied to microtiter plates containing indicator cells which are easy to identify.
• the cell lysis changes the morphology of the cells. This cell lysis is easy to measure under the microscope.
• the viruses tested, coated and uncovered, were added in high concentration to a 0.01NH 2 SO 4 solution (pH 2, 30 ° C), which also contains TSO. After 10 min, 3 h and 72 h, the activity of the viruses was determined. After 10 min. were the enveloped viruses PRV and MuLV, after 3 h the non-enveloped viruses FCV and PPV were no longer infectious. The use of a counter sample neutralized with 1 N NaOH also showed, after 3 h of incubation, that all 4 viruses were still virulent. Because of the cytotoxicity of a 0.2% potassium dichromate solution in 0.01 N sulfuric acid, testing this solution as a comparison was not possible.
• the embryo coagulation coefficient could be obtained.
• the embryo-coagulation coefficient is a clear marker for the survivability of worm egg suspensions.
• the use of preservatives enabled the inactivation of contaminating microorganisms even at pH 4. Benzoic acid, sorbic acid, its salts and propylene glycol have proven to be preferred orally acceptable preservatives (see Example 4 a - e).
• the samples tested contained 10,000 TSO / ml ( ⁇ 10%) in pH 4 phosphate buffer and either 0.2% benzoic acid, 0.1% sorbic acid or 15% propylene glycol.
• Pseudomonas aruginosa Staphylococcus aureus, Candida albicans and
• sorbic acid, benzoic acid and / or p-hydroxybenzoic acid esters are preferably used or propylene glycol combined with these preservatives is used.
• a further advantage of the combined inactivation of microorganisms by means of dilute acids and the use of preservatives is that a possible recontamination with new germs can be prevented during the further use of the TSO suspension. The growth of new germs is avoided by the described combined method of inactivating and preserving a TSO suspension.
• Preparation for further preparation and use in the patient requires raising the pH to a range of pH 2-7, preferably pH 4-6. Addition of suitable preservatives is also required for this third step. Preservatives to be used must be suitable for oral use. Sorbic acid in a concentration of 0.1-0.2%, benzoic acid in a concentration of 0.1-0.3%, p-hydroxybenzoic acid ester in concentrations of 0.02-0.3%, have proven suitable here. or propylene glycol in a 5-20% aqueous solution. Also suitable are mixtures of the above-mentioned preservatives.
• Example 5 Viability of worm eggs
• Non-embryonated worm eggs at a concentration of 40'0OO eggs / ml were suspended in different media and embryonated (22-25 ° C). After an incubation period of 60 and 90 days, the individual suspensions were examined microscopically and the embryonation coefficient was calculated from the total number of worm eggs and the number of morphologically intact and embryonated worm eggs.
• the embryo coefficient (EK) is an indicator of the viability of worm eggs. At the beginning of embryonation, only non-embryonic worm eggs are present, so that the coefficient is zero. It increases during the duration of embryonation and reaches its maximum at day 60 under the selected incubation conditions. As a rule, values of around 90% are achieved. Suspension medium EK day 0 EK day 60 EK day 90
• the preparation for further preparation requires raising the pH to a range of pH 2-7, preferably pH 4-6, as well as the addition of suitable preservatives.
• Preservatives to be used must be suitable for oral use. Sorbic acid in a concentration of 0.01-0.2%, benzoic acid in a concentration of 0.1-0.3%, or propylene glycol in a 5-20% strength aqueous solution have proved to be suitable. Also suitable are mixtures of the above-mentioned preservatives.

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