Fachbereich Veterinärmedizin



    Therapeutischer Nutzen der Anwendung einer CpG-ODN GNP-Inhalationslösung zur Behandlung der COB-Erkrankung des Pferdes:
    eine klinische Feldstudie, einschließlich der Erhebung allgemeiner statistischer Daten zur COB-Erkrankung in Deutschland anhand eines Fragenbogens (2013)

    Lehmann, Beatrice (WE 17)
    Berlin: Mensch und Buch Verlag, 2013 — XIV, 213 Seiten
    ISBN: 978-3-86387-404-9
    URL (Volltext): http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000095828
    Klinik für Pferde, allgemeine Chirurgie und Radiologie

    Oertzenweg 19 b
    14163 Berlin
    +49 30 838 62299

    Abstract / Zusammenfassung

    Recurrent airway obstruction (RAO) is the most frequently occurring respiratory disease of horses in the northern hemisphere. It is based on the interaction of certain genetic and environment-associated factors and, therefore, occurs in affected horses only after contact with allergens. As for its pathology, RAO of the horse is comparable to asthma in humans. In serious cases, RAO leads to an enormous psychological strain for the affected patients and also constitutes an extraordinarily great emotional and financial burden for the horse owners. Thus, highest priority should be given to the further investigation of etiology, pathology and long-term therapeutic possibilities.
    The purpose of the present study was, on the one hand, to collect general data about RAO in Germany with the aid of a questionnaire homepage and to acquire patients for the clinical field study performed subsequently. On the other hand, the effectiveness and tolerance of an inhalative administration of cytosine-phosphateguanine-oligodeoxynucleotides (CpG-ODN), bonded to a nanoparticle carrier system (Gelantin Nanoparticels, GNP), was tested in a double-blinded, prospectively randomized and placebo-controlled field study as a new therapeutic procedure for treatment of RAO in the horse. The questionnaire homepage was developed in cooperation with the computer operation group of the LMU Munich using the LimeSurvey® program. With help of the homepage, data was collected regarding the first occurrence of RAO, various courses of the disease, possible variation of symptoms, the development of each patient’s performance, the purchase and sale of RAO -affected horses, and the type of diagnosis and therapy carried out by the treating veterinarians. In total, 266 completely filled out questionnaires could be evaluated. Analysis of the questionnaires showed that the mean age for first occurrence of RAO in Germany was 10.10 years (SE = 0.297), and that exacerbation of the disease occurred in 70% of cases. In these cases, the symptoms of the disease continued for about eight weeks. The contact to dry hay was determined to be the trigger for exacerbation of the disease in over half of all cases (52.3%). The temporal interval between two exacerbations, calculated on the basis of the survey’s result, averaged 8 months.
    By means of our own analysis, it could be proven that the number as well as the extent of the occurring symptoms of the disease clearly increased over the course of the disease. The symptoms “enforced abdominal breathing” (increase by 16.5%), “reduced performance” (increase by 12.8%) and “coughing at beginning of riding” (increase by 10.5%) showed the greatest increase in the context of exacerbations of RAO.
    In over half of the cases (65%), the horses’ performance clearly decreased according to information from the owners. Provided that a participation in horse shows took place at all after the occurrence of RAO, a decrease of achieved success of about ten percent was observed. An amount of 856 € (SE = 65,9) was calculated as average annual additional costs for the horse owners as a result of their horse’s RAO. Presumably, all of these ascertained negative factors as a whole led to the result that in 71.4% of cases a new purchase of a RAO-affected horse was categorically rejected by the horse owners.
    The results of the questionnaire showed that the chosen medium was accepted well by the horse owners and that, by using a questionnaire homepage, it was very easy to collect study-related data regarding veterinarian questions. The creation of a homepage with which the success of a therapeutic method can be proven or which helps for diagnosis of diseases could represent a meaningful further development of the questionnaire homepage used in this study. In summary, the result of our own survey shows that RAO of the horse in Germany is a chronic progressive disease, in which in case of exacerbations symptoms of the disease in partially severe form remain in the affected patients over a period of two months. The investigation of therapeutic options which result in a long-term improvement of these symptoms in RAO-affected horses should therefore be granted highest priority.
    The CpG-ODN/GNP inhalation solution, used within the scope of the second part of this study, represents a new and promising therapeutic approach with possible long-term effect for treatment of RAO in horses. By use of the CpG-ODN/GNP inhalation solution, an immunomodulatory effect (“Th1-Shift”) was supposed to be induced in the patients in the field study. This effect had already been proven in previous in-vitro and animal- experimental in-vivo studies. Since the effect of CpG-ODN is less pronounced in large mammals (than as, e.g., in rodents), gelatin nanoparticles were used as carrier system for the CpG-ODN. These protected the CpG-ODN against a premature decomposition through the body’s nucleases and led to an enhancement of the effect in the target cells.
    A total of 24 horses were admitted to the clinical field study, whose owners had applied for acceptance of their horse to the study through the questionnaire homepage or who were referred by their general veterinarian practitioners. In each patient the current severity of the disease (“mild-moderate” or “severe”) was initially determined on the basis of an established scoring system. In consideration of the previously determined “disease score”, an allocation to the verum group or the placebo group was carried out by lot procedure. The placebo group consisted of 4 mildly to moderately affected patients and 4 severely affected patients; likewise, the verum group consisted of 8 mildly to moderately affected patients and 8 severely affected patients. The patients of both groups were inhaled five times (at intervals of two days each), whereas the patients of the verum group were administered the CpGODN/GNP inhalation solution (0.18mg CpG-ODN bound to 3.75mg GNP, dissolved in 2.5ml highly purified water). The patients of the placebo group were inhaled only with highly purified water and GNP, whereby the share of CpGODN in the inhalation solution was completely replaced by highly purified water. After completion of the inhalation therapy, first and second follow-up examinations were performed (in each case one day after the last inhalation treatment and again after four weeks) to be able to prove possible therapeutic results directly following the treatment as well as after a greater temporal interval. The goal of the practical study was to prove the positive effect of the used inhalation solution based on various clinical parameters (respiratory rate at rest, breathing type, nasal discharge, results of the auscultation of the airways, arterial blood gas analysis, measurement of intrapleural pressure) as well as endoscopically determinable parameters (amount of secretion, viscosity of secretion, assessment of the tracheal bifurcation) and a cytological examination of tracheobronchial secretion.
    No local or systemic intolerance could be detected in any of the patients during or after the inhalation therapy, which speaks for good tolerance of the used dose rate and the used inhalation protocol. Within both placebo groups (mild-moderate and severely affected patients), no significant improvements could be shown for any of the examined parameters. Above all, the results within the placebo groups prove the good tolerance of the solely inhalative application of GNP in the horse. In both verum groups, however, after five inhalations with the CpG-ODN/GNP inhalation solution, a significant improvement of respiratory rate and oxygen partial pressure could be observed.
    The average endoscopically determined amount of tracheobronchial secretion decreased significantly in the group of mildly to moderately affected horses after five inhalations. Also, a significant long-term effect could be proven for this reduction. In the group of severely affected patients, a significant decrease in the amount of secretion could be observed, that also lasted over a period of four weeks.
    At the first follow-up examination, the apparent breathing type in the verum group of mildly to moderately affected horses was very significantly improved and in the verum group of severely affected horses highly significantly improved. In both verum groups, a significant long-term effect of this finding could be proven.
    Clear indications for the existence of a hypoxic metabolic situation are the increased respiratory breathing rate as well as the decrease in the arterially evaluated oxygen partial pressure. Hence, the synchronic improvement of both examined parameters showed that, with only five inhalations with the CpGODN/GNP inhalation solution, the existing dysfunction of pulmonary gas exchange in RAO patients was significantly improved.
    In addition, one can assume that, through the reduction of the amount of secretion in the airways, the often excruciating coughing symptoms could be reduced clearly and on a long-term basis.
    The unphysiological breathing type, detected at the beginning of therapy, was a sign for increased maximum tidal volume difference (ΔPplmax) that is attributed to the bronchoconstriction which always exists in RAO patients. The improvement in the breathing type shown by the patients could, therefore, also be seen as evidence for a decrease of the existing bronchoconstriction. Consequently, one of the three main pathomechanisms of RAO could be treated most successfully and with development of a long-term effect through a mere five-time application of the CpG-ODN/GNP inhalation solution. Equivalent long-term effects have not been achieved to date (to the author’s knowledge) with any other therapeutic method without a simultaneous change in housing conditionF In closing, it should be noted that the present study provides proof of the practicability of inhalation therapy with CpG-ODN/GNP inhalation solutions under field conditions and their therapeutic effectiveness. In particular, it should be emphasized that in the therapeutic protocol carried out here a positive longterm effect could be achieved in seven of the ten examined parameters (breathing type, nasal discharge, results of the auscultation, amount of secretion, viscosity of secretion, appearance of the tracheal bifurcation and the number of neutrophil granulocytes in the total cell count of the tracheobronchial secretion in severelyaffected patients).
    Therefore, the immunologic mechanism (“Th1-shift”), on which the therapeutic effect of the used inhalation solution is based, should be investigated more precisely in future studies to be able to accelerate an enhancement or extension of the therapeutic effects.
    The application of a CpG-ODN/GNP inhalation solution in combination with the specific immunotherapy (SIT) could lead to a further enhancement of the positive effects and should be investigated in future studies.