Insights from an Alternative Administration Procedure in the Göttingen Minipig: When Innovation Meets Expertise

Intravesical drug delivery (IDD), involving placement of drugs directly into the bladder through a urethral catheter, is commonly used to treat various bladder diseases including bladder cancers, urinary tract infections and interstitial cystitis. This regional therapy thus provides a high concentration of drugs to the diseased bladder, with low risk of systemic side effects. Here, we describe the use of Göttingen Minipig as a model for the assessment of local tolerance/toxicity, following repeated intravesical instillation of new drugs. Although challenging to perform and requiring significant technical skills, the procedure was shown to be overall safe and well tolerated.

Introduction

The minipig experimental model has become increasingly used in the last few years, as an alternative to the more traditional dog and nonhuman primate (NHP) non-rodent species (1). Indeed, when compared to other non-rodent species, the closer correlation with humans from anatomical, physiological and biochemical points of view makes the minipig model an excellent candidate for translational research (2). Specifically, pigs appear to be a more suitable model than dogs for immunological evaluation (3) and they have been used in investigations on cardiovascular and respiratory safety pharmacology (4, 5), as well as in studies of dermal (6), neurological, reproductive and other different pathologies (7, 8). Besides, their relative small size allows easier handling procedures, together with a reduction in the amount of food, housing space and test item needed to carry out a study. This model is therefore generally accepted by regulatory authorities, provided it is adequately justified (9). Although several breeds of minipigs have been produced, among these, Göttingen Minipigs are the most used for biomedical purposes (10, 11). 

The possibility to perform and exploit almost any type of route of administration, constitutes another important feature of this non-clinical model that reinforces its translational power (12). In this regard, besides common routes of administration (e.g. oral, intravenous, subcutaneous, dermal and intramuscular), other unusual ones can be exploited to perform toxicology studies in minipigs, including intravitreal, intratracheal, intraarticular, intranasal and intravesical (13, 14).

Specifically, intravesical drug delivery (IDD), providing a high concentration of drugs to the diseased bladder, represents a viable approach for treating various bladder diseases including bladder cancers, urinary tract infections and interstitial cystitis (15, 16). Indeed, owing to its anatomical features, urinary bladder represents an effective barrier that prevents the diffusion of toxic substances from the urine into the bloodstream, this reducing the risk of systemic side effects after local drug administration (17). 

Due to the similarity to humans with respect to the urogenital anatomy, urine density, and immune system physiology (18), pig has proved to be a suitable model for non-clinical studies involving IDD. Importantly, pigs are natural host of uropathogenic Escherichia coli (UPEC), the predominant etiologic agent of human urinary tract infections (UTIs) (14, 19). 

Here we present a detailed protocol for tolerance/toxicity studies after repeated urinary bladder instillation in Göttingen Minipigs. Only female minipigs are used since a retrograde approach to the bladder through the urethra is not possible in males due to their penile anatomy (sigmoid flexure of the penis).

Materials and methods

Six Göttingen Minipig females, approximately 4-5 months old and weighing 9-11 kg, were purchased from Ellegaard Göttingen Minipigs (Dalmose, Denmark), as part of a batch of 26 female Göttingen minipigs, to be used in a repeated dose toxicity study. These animals acted as controls in a regulatory toxicity study carried out at European Research Biology Center (ERBC) S.r.l., (Pomezia, Rome, Italy), a GLP certified facility. Procedures and facilities were compliant with the requirements of the Directive 2010/63/EU on the protection of animals used for scientific purposes. The national transposition of the Directive is defined in Decreto Legislativo 26/2014. ERBC is fully accredited by Association for Assessment and Accreditation of Laboratory Animal Care International and the aspects of the protocol concerning animal welfare were approved by the animal welfare body. 

The animals were group housed (two animals per pen) in indoor enclosures of approximately 200×135 cm floor size both during the acclimatization period and experimental phases. Single housing was limited to the periods necessary for a correct performance of animals care and experimental procedures. Drinking water was offered ad libitum throughout the study, and a weighed amount of diet [Altromin 9069, Maintenance diet for minipig, Altromin Spezialfutter GmbH & Co. KG ImSeelenkamp 20, 32791 Lage, Germany] was offered daily divided in two rations.

Animals, fasted for about 12 hours before administration, were premedicated with a mixture of Ketamine (10 mg/kg, intramuscular), Xylazine (1 mg/kg, intramuscular) and Diazepam (5-10 mg/kg, intravenous, if necessary) and maintained by Isoflurane (1.5-3%) inhalation (Oxygen flow 1.5-2 L/min) (Fig. 1). Following the pre-anesthesia, each animal was subjected to gaseous anesthesia by means of a mask (2-5% Isoflurane), then intubated through an orotracheal tube and maintained under gaseous anesthesia (1.5-3% Isoflurane) for the whole treatment period (approximately 1-hour exposure). The anaesthetized animal was placed onto the operating table in sternal recumbency. Once the desired stage of anesthesia was achieved, the perineum and vulva were washed and cleaned thoroughly with septal scrubs and a pre-perforated surgical drape was placed. The Foley catheter (Fig. 2), embedded with lubricant gel, was gently introduced into the urethra until the urine flowed into it. A speculum was used to facilitate the introduction of the catheter. The balloon was inflated with approximately 5 mL of sterile saline solution in order to avoid accidental removal of the catheter. When all the urine was removed by gravity, the animal was dosed. A total of 100 mL of sterile physiological saline (negative control) was introduced through the catheter into the urinary bladder followed by 1.5 mL of sterile physiological saline and left in place for an exposure period of 1 hour. At the end of the exposure period, the Foley catheter was removed after the emptying of the urinary bladder. Animals were dosed once weekly for a total of four times, on Days 1, 8, 15 and 22 of the study (4 weeks). Animals in the main phase (4 females) were euthanized the day after last dosing. A recovery period of 2 weeks was allowed after last administration for 2 females. 

In vivo observations including daily clinical signs, body weight, food and water consumption, physical examination, electrocardiography (ECG) and clinical pathology investigations (including urinalysis) were carried out throughout the observation period.

Immediately after autopsy, tissue samples for histopathological examination of the urinary bladder and urethra were collected. The urinary bladder was opened in the dorsal region from the urethra, placed on a rigid cardboard and pinned at its borders, to keep it flat during formalin fixation.

Six formalin-fixed samples were taken (Fig. 3): 3 in the urinary bladder at the middle (ventral) position in the apical, middle and caudal area (Levels 1, 2, 3) and 3 in the urethra apical, middle and caudal area (Levels 4, 5, 6). Tissues were embedded in paraffin wax, the blocks were sectioned at 5 µm thickness and routine hematoxylin and eosin (H&E) staining was performed. A microscopic examination was performed by a board-certified veterinary pathologist, on the full list of tissues including the urinary tract (kidneys, ureters, urinary bladder, and urethra) for main animals and on the urinary bladder and urethra for recovery animals. The scoring of the lesions was done semi-quantitatively, using a 5-point grading scale for all reported microscopic findings (1= minimal, 2= mild, 3= moderate, 4= marked, 5= severe).

Results and discussion

All minipigs evaluated in this study were considered healthy on the basis in vivo observations, including body weight, food and water consumption, physical examination, clinical pathology analyses, and ECG. There were no relevant changes of parameters evaluated at the end of the dosing and recovery periods compared to pre-dose results. 

At necropsy, there were no macroscopic observations in the urinary bladder and urethra at the end of the dosing period. At the end of the recovery period, a single pale area was noted in the urinary bladder of one animal but in the absence of microscopic correlates, it was not considered to be relevant.

Procedure-related microscopic findings were observed in the urinary bladder and urethra. At the end of the dosing period, they consisted of minimal or mild, multifocal mixed inflammatory cell infiltrates in the urothelium and lamina propria of the urinary bladder of 2/4 animals (Fig. 4) and/or urethra of 3/4 animals (Fig. 5), and minimal focal hemorrhage in the urinary bladder or urethra (2/4 animals). Mixed cell infiltrates, affecting all levels examined in both tissues, were composed of mononuclear cells and neutrophils in variable proportion, with higher numbers of neutrophils present in the urethra compared to urinary bladder. A single animal did not show any inflammatory infiltrate in the urinary bladder or urethra. The few microscopic findings noted in the kidneys of main animals were common background findings for the minipig and were not considered to be related to the procedure (20). There were no microscopic findings in the ureters. At the end of the recovery period, minimal or mild, multifocal mixed inflammatory cell infiltrates were still observed in both animals, in the urinary bladder only, in association with minimal multifocal urothelial hyperplasia in one of them. There was full reversibility of the microscopic changes in the urethra.

Results obtained in this study demonstrate that the Göttingen Minipig is an excellent non-rodent animal model for the assessment of local tolerance/toxicity, following repeated (once per week and up to four weeks) intravesical instillation of new drugs. The multiple sampling in specific anatomic regions of the urinary bladder allowed proper histopathological evaluation of any potential findings related to the treatment procedure. Low grade inflammation, hemorrhage and/or reactive urothelial hyperplasia in the urinary bladder and urethra were observed and were considered secondary to repeated microtrauma of the urinary mucosa during catheterization. This points out the need of incorporating negative controls in toxicological studies using this route of administration in order to differentiate potential histopathological changes induced by test items or placebo from procedure-related effects.

On the whole, these results provide evidence for the use of the intravesical administration procedure as safe and well tolerated in this animal model, with procedure-related changes detectable in the urinary bladder and urethra at the microscopic level only. While the procedure is safe and well tolerated, it is challenging to perform and involves significant technical skills by the veterinary staff performing it.

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