The Non-Human Primate in Safety Assessment of a Bifunctional Long-Acting Insulin Analogue.

Species selection plays a pivotal part during non-clinical safety assessment in drug development. If possible, use of non-human primates (NHPs) should be avoided due to ethical considerations. However, limiting factors as lack of pharmacologic activity in other species could necessitate use of NHPs. LAI-PCSK9i is a bi-functional molecule combining a long-acting insulin analogue with a PCSK9 inhibitor peptide aiming to provide glycaemic control and to reduce plasma LDL concentrations. The NHP was chosen for the safety assessment of LAI-PCSK9i being the most relevant species with basal levels and plasma lipid composition closest to humans, while the dog and initially also the minipig were deemed irrelevant due to lack of pharmacologic activity on LDL-lowering and biological differences in lipid profiles. An in vivo tolerability and toxicokinetic study of LAI-PCSK9i in NHPs showed recurrent and severe hypoglycaemia at very low doses. Therefore, the minipig was re-evaluated and a follow-up study thoroughly assessing blood glucose and cholesterol levels and clinical signs illustrated that minipigs dosed with LAI-PCSK9i, tolerated the compound and LAI-PCSK9i decreased glucose and LDL over time. This work underlines that careful consideration is required when selecting species during safety assessment in drug development. The tolerability issue in NHPs led to the subsequent selection of the minipig for safety evaluation of LAI-PCSK9i although as a suboptimal alternative, which unexpectedly had a measurable pharmacologic response on LDL lowering. In conclusion, the NHPs may be unsuitable as test species for safety assessment of long-acting insulin analogues due to high sensitivity to recurring hypoglycaemic episodes.

Noninvasive assessment of pulse-wave velocity and flow-mediated vasodilation in anesthetized Göttingen minipigs.

Few methods for noninvasive assessment of arterial stiffness and endothelial dysfunction in porcine models are available. The aim of this study was to evaluate methods for assessment of arterial stiffness and endothelial dysfunction in anesthetized Göttingen minipigs. Pulse-wave velocity (PWV) was assessed in male Göttingen minipigs (n = 8; age approximately 60 wk) by using applanation tonometry of the carotid and femoral arteries. In addition, flow-mediated vasodilation (FMD) was assessed by using vascular ultrasonography of the brachial artery to evaluate endothelial dysfunction. To evaluate the reproducibility of the methods, minipigs were anesthetized by intravenous infusion of ketamine and midazolam and examined every other day for a total of 3 trials. Neither examination day nor systolic, diastolic, or mean arterial blood pressure statistically influenced PWV or FMD. The median interexamination coefficient of variation was 17% for PWV and 59% for FMD. Measured values of PWV corresponded largely to those in clinically healthy humans, but FMD values were lower than expected for lean, young animals. Although the ketamine-midazolam anesthesia we used has been associated with minor hemodynamic effects in vivo, in vitro studies suggest that both drugs are vasodilatory. Therefore anesthesia might have influenced the endothelial response, contributing to the modest FMD response and the concurrent high coefficients of variation that we noted. We conclude that PWV­but not FMD­showed acceptable interexamination variation for its potential application in porcine models.

Functional network analysis of obese and lean Göttingen minipigs elucidates changes in oxidative and inflammatory networks in obese pigs.

The Göttingen minipig model of obesity is used in pre-clinical research to predict clinical outcome of new treatments for metabolic diseases. However, treatment effects often remain unnoticed when using single parameter statistical comparisons due to the small numbers of animals giving rise to large variation and insufficient statistical power. The purpose of this study was to perform a correlation matrix analysis of multiple multi-scale parameters describing co-segregation of traits in order to identify differences between lean and obese minipigs. More than 40 parameters, ranging from physical, cardiovascular, inflammatory and metabolic markers were measured in lean and obese animals. Correlation matrix analysis was performed using permutation test and bootstrapping at different levels of significance. Single parameter comparisons yielded significant differences between lean and obese animals mainly for known physical traits. On the other hand, functional network analysis revealed new co-segregations, particularly in the domain of inflammatory and oxidative stress markers in the obese animals that were not present in the lean. Functional networks of lean or obese minipigs could be utilised to assess drug effects and predict changes in parameters with a certain degree of precision, on the basis of the networks confidence intervals. Comparison of functional networks in minipigs with those of human clinical data may be used to identify common parameters or co-segregations related to obesity between animal models and man.

Development of left ventricular hypertrophy in a novel porcine model of mitral regurgitation.

OBJECTIVES: We aimed to develop a porcine model for chronic nonischemic mitral regurgitation (MR) to investigate left ventricular (LV) enlargement and eccentric hypertrophy. DESIGN: Nonischemic MR was induced in 30 pigs by open-chest immobilization of the posterior mitral leaflet by transannular traction sutures that where applied in transmyocardial fashion. A sham operated control group (n = 13) was included. Echocardiographic LV size and heart weight assessed at euthanasia were used to evaluate the development of LV enlargement and eccentric hypertrophy after 8 weeks follow-up. RESULTS: Eight pigs died and seven were excluded due to mediastinal infection (n = 2) or failure to produce MR (n = 5). Thus, 28 pigs were included and were divided into three groups: controls (n = 12), mild MR (mMR; n = 10), and moderate to severe MR (sMR; n = 6). The change in LV internal diameter in diastole (LVIDd) from baseline to follow-up was significantly higher in the sMR group compared to that of the control group (P = 0.0017). Furthermore, LV weight was significantly increased in the mMR (P = 0.047) and the sMR (P = 0.0087) groups compared to that of the control group. CONCLUSIONS: A new model for chronic moderate to severe nonischemic MR with development of LV enlargement and eccentric hypertrophy within 8 weeks has been established in pigs.

Plasma proANP and SDMA and microRNAs are associated with chronic mitral regurgitation in a pig model.

OBJECTIVE: NON-ISCHEMIC MITRAL REGURGITATION (MR) IS PRIMARILY CAUSED BY MYXOMATOUS MITRAL VALVE (MV) DISEASE LEADING TO ADAPTIVE REMODELING, ENLARGEMENT, AND DYSFUNCTION OF THE LEFT VENTRICLE. THE AIM OF THIS STUDY WAS TO EXAMINE THE REGULATION OF PLASMA MARKERS AND SEVERAL CARDIAC KEY GENES IN A NOVEL PORCINE MODEL OF NON-ISCHEMIC MR. METHODS AND RESULTS: Twenty-eight production pigs (Sus scrofa) were randomized to experimental MR or sham surgery controls. MR was induced by external suture(s) through the posterior MV leaflet and quantified using echocardiography. The experimental group was subdivided into mild MR (mMR, MR=20-50%, n=10) and moderate/severe MR (sMR, MR >50%, n=6) and compared with controls (CON, MR ≤10%, n=12). Eight weeks postoperatively, follow-up examinations were performed followed by killing. Circulating concentrations of pro-atrial natriuretic peptide (proANP), l-arginine, asymmetric dimethylarginine, and symmetric dimethylarginine (SDMA) were measured. MV, anterior papillary muscle, and left ventricular free wall tissues were collected to quantify mRNA expression of eNOS (NOS3), iNOS (NOS2), MMP9, MMP14, ANP (NPPA), BNP (NPPB), and TGFB1, 2, and 3 and five microRNAs by quantitative real-time PCR. Pigs with sMR displayed markedly increased plasma proANP and SDMA concentrations compared with both controls and mMR (P<0.05). The expression of all genes examined differed significantly between the three localizations in the heart. miR-21 and miR-133a were differently expressed among the experimental groups (P<0.05). CONCLUSIONS: Plasma proANP and SDMA levels and tissue expression of miR-21 and miR-133a are associated with severity of chronic MR in an experimental porcine model.

Characterisation of gut microbiota in Ossabaw and Göttingen minipigs as models of obesity and metabolic syndrome.

BACKGROUND: Recent evidence suggests that the gut microbiota is an important contributing factor to obesity and obesity related metabolic disorders, known as the metabolic syndrome. The aim of this study was to characterise the intestinal microbiota in two pig models of obesity namely Göttingen minipigs and the Ossabaw minipigs. METHODS AND FINDINGS: The cecal, ileal and colonic microbiota from lean and obese Osabaw and Göttingen minipigs were investigated by Illumina-based sequencing and by high throughput qPCR, targeting the 16S rRNA gene in different phylogenetic groups of bacteria. The weight gain through the study was significant in obese Göttingen and Ossabaw minipigs. The lean Göttingen minipigs' cecal microbiota contained significantly higher abundance of Firmicutes (P<0.006), Akkermensia (P<0.01) and Methanovibribacter (P<0.01) than obese Göttingen minipigs. The obese Göttingen cecum had higher abundances of the phyla Spirochaetes (P<0.03), Tenericutes (P<0.004), Verrucomicrobia (P<0.005) and the genus Bacteroides (P<0.001) compared to lean minipigs. The relative proportion of Clostridium cluster XIV was 7.6-fold higher in cecal microbiota of obese Göttingen minipigs as compared to lean. Obese Ossabaw minipigs had a higher abundance of Firmicutes in terminal ileum and lower abundance of Bacteroidetes in colon than lean Ossabaw minipigs (P<0.01). Obese Ossabaws had significantly lower abundances of the genera Prevotella and Lactobacillus and higher abundance of Clostridium in their colon than the lean Ossabaws. Overall, the Göttingen and Ossabaw minipigs displayed different microbial communities in response to diet-induced obesity in the different sections of their intestine. CONCLUSION: Obesity-related changes in the composition of the gut microbiota were found in lean versus obese Göttingen and Ossabaw minipigs. In both pig models diet seems to be the defining factor that shapes the gut microbiota as observed by changes in different bacteria divisions between lean and obese minipigs.

Orosomucoid expression profiles in liver, adipose tissues and serum of lean and obese domestic pigs, Göttingen minipigs and Ossabaw minipigs.

The acute phase protein orosomucoid (ORM) has anti-inflammatory and immunomodulatory effects, and may play an important role in the maintenance of metabolic homeostasis in obesity-induced low-grade inflammation. Even though the pig is a widely used model for obesity related metabolic symptoms, the expression of ORM has not yet been characterized in such pig models. The objective of this study was to investigate the expression of ORM1 mRNA in liver, visceral adipose tissue, subcutaneous adipose tissue (SAT) from the abdomen or retroperitoneal abdominal adipose tissue (RPAT) and SAT from the neck, as well as the serum concentration of ORM protein in three porcine obesity models; the domestic pig, Göttingen minipigs and Ossabaw minipigs. No changes in ORM1 mRNA expression were observed in obese pigs compared to lean pigs in the four types of tissues. However, obese Ossabaw minipigs, but none of the other breeds, showed significantly elevated ORM serum concentrations compared to their lean counterparts. Studies in humans have shown that the expression of ORM was unchanged in adipose tissue depots in obese humans with an increased serum concentration of ORM. Thus in this respect, obese Ossabaw minipigs behave more similarly to obese humans than the other two pig breeds investigated.

Circulating cytokine concentrations in dogs with different degrees of myxomatous mitral valve disease.

Cytokines have been associated with the progression of congestive heart failure (CHF) in humans and may be implicated in the pathophysiology of myxomatous mitral valve disease (MMVD) in dogs. The aim of this study was to determine the serum concentrations of cytokines in dogs with MMVD. The study included 16 Cairn terriers with no or minimal mitral regurgitation (MR), 41 Cavalier King Charles Spaniels (CKCS) with different degrees of MR and 11 dogs of different breeds with CHF due to MMVD. Granulocyte-macrophage colony-stimulating factor, interferon-γ, interleukin (IL)-2, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, keratinocyte-derived chemokine, interferon-γ-induced protein and monocyte chemoattractant protein-1 (MCP-1) were measured using a canine-specific multiplex immunoassay. CHF dogs had significantly higher MCP-1 concentrations than dogs with no or minimal MR. Among the CKCS, IL-2 and IL-7 decreased with increasing left atrial size and IL-7 also decreased with increasing MR. IL-8 decreased with increasing left ventricular end-systolic internal dimensions. MCP-1 was increased in CHF dogs compared to healthy control dogs and IL-2, IL-7 and IL-8 decreased with increasing indices of disease severity. The results suggest a role for these cytokines in canine MMVD and CHF.

Brain-natriuretic peptide and cyclic guanosine monophosphate as biomarkers of myxomatous mitral valve disease in dogs.

Elevations in the plasma concentrations of natriuretic peptides correlate with increased severity of myxomatous mitral valve disease (MMVD) in dogs. This study correlates the severity of MMVD with the plasma concentrations of the biomarkers N-terminal fragment of the pro-brain-natriuretic peptide (NT-proBNP) and its second messenger, cyclic guanosine monophosphate (cGMP). Furthermore, the L-arginine:asymmetric dimethylarginine (ADMA) ratio was measured as an index of nitric oxide availability. The study included 75 dogs sub-divided into five groups based on severity of MMVD as assessed by clinical examination and echocardiography. Plasma NT-proBNP and cGMP concentrations increased with increasing valve dysfunction and were significantly elevated in dogs with heart failure. The cGMP:NT-proBNP ratio decreased significantly in dogs with heart failure, suggesting the development of natriuretic peptide resistance. Although the l-arginine:ADMA ratio decreased with increasingly severe MMVD, this was largely due to the older age of the dogs with heart failure.

Predictive value of natriuretic peptides in dogs with mitral valve disease.

Natriuretic peptides are useful in diagnosing heart failure in dogs. However, their usefulness in detecting early stages of myxomatous mitral valve disease (MMVD) has been debated. This study evaluated N-terminal (NT) fragment pro-atrial natriuretic peptide (NT-proANP) and NT-pro-brain natriuretic peptide (NT-proBNP) in 39 Cavalier King Charles Spaniels (CKCS) with pre-clinical mitral valve regurgitation (MR), sixteen dogs with clinical signs of heart failure (HF) and thirteen healthy control dogs. Twenty seven CKCS and ten control dogs were re-examined 4 years after the initial examination and the status of the dogs 5 years after the initial examination was determined by telephone calls to the owner. All dogs were evaluated by clinical examination and echocardiography. CKCS with severe MR had higher NT-proANP and NT-proBNP compared to controls and CKCS with less severe MR. Dogs with clinical signs of HF had markedly elevated NT-proANP and NT-proBNP. Plasma concentrations of the natriuretic peptides measured at re-examination could predict progression in regurgitant jet size.

Increased nitric oxide release and expression of endothelial and inducible nitric oxide synthases in mildly changed porcine mitral valve leaflets.

BACKGROUND AND AIM OF THE STUDY: Little is known of the local role of nitric oxide (NO) in heart valves in relation to heart valve diseases. The study aim was to examine NO release and the expression of both endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in relation to early local changes in porcine mitral valves. METHODS: A histological evaluation of mitral valve leaflets from slaughter pigs and sows was made, and the expression of eNOS and iNOS protein measured using immunohistochemistry. Furthermore, mRNA levels of eNOS and iNOS were measured using real-time RT-PCR. A calibrated NO-specific electrode was used to measure local NO release in specific regions of the anterior mitral leaflet from slaughter pigs and sows interchordally at the tip of the leaflet (region A), at the chordal insertion (region B), and at the center of the leaflet (region C). RESULTS: Leaflets from sows had an increased accumulation of mucopolysaccharides (MPS) compared to those from slaughter pigs. Furthermore, mRNA levels of eNOS and iNOS were significantly increased in region C due to very high levels of expression in some sow leaflets. NO release in the sow mitral valve leaflet was increased in regions B and C compared to that in region A. CONCLUSION: The relative distribution of NO release is increased in regions of porcine mitral valve leaflets with deposition of MPS and defraction of the valve structure, which may reflect changes in both eNOS and iNOS expression.

Sensory nerve inactivation by resiniferatoxin improves insulin sensitivity in male obese Zucker rats.

Recent studies have suggested that sensory nerves may influence insulin secretion and action. The present study investigated the effects of resiniferatoxin (RTX) inactivation of sensory nerves (desensitization) on oral glucose tolerance, insulin secretion and whole body insulin sensitivity in the glucose intolerant, hyperinsulinemic, and insulin-resistant obese Zucker rat. After RTX treatment (0.05 mg/kg RTX sc given at ages 8, 10, and 12 wk), fasting plasma insulin was reduced (P < 0.0005), and oral glucose tolerance was improved (P < 0.005). Pancreas perfusion showed that baseline insulin secretion (7 mM glucose) was lower in RTX-treated rats (P = 0.01). Insulin secretory responsiveness to 20 mM glucose was enhanced in the perfused pancreas of RTX-treated rats (P < 0.005) but unaffected in stimulated, isolated pancreatic islets. At the peak of spontaneous insulin resistance in the obese Zucker rat, insulin sensitivity was substantially improved after RTX treatment, as evidenced by higher glucose infusion rates (GIR) required to maintain euglycemia during a hyperinsulinemic euglycemic (5 mU.kg(-1).min(-1)) clamp (GIR(60-120min): 5.97 +/- 0.62 vs. 11.65 +/- 0.83 mg.kg(-1).min(-1) in RTX-treated rats, P = 0.003). In conclusion, RTX treatment and, hence, sensory nerve desensitization of adult male obese Zucker rats improved oral glucose tolerance by enhancing insulin secretion, and, in particular, by improving insulin sensitivity.

Effects of high-fat feeding and fasting on ghrelin expression in the mouse stomach.

Ghrelin is a peptide identified as an endogenous ligand for the growth hormone secretagogue receptor. Studies have shown that ghrelin stimulates growth hormone, promotes food intake and decreases energy expenditure. Furthermore, feeding status seems to influence plasma ghrelin levels, as these are increased during fasting, whereas feeding and oral glucose intake reduce plasma ghrelin. This study examined whether standardized obesity and fasting affect cellular expression of ghrelin. Specimens from the gastrointestinal tract of fed or 18-h fasted, low-fat or high-fat fed (10 weeks on diet) C57BL/6J mice were studied by immunocytochemistry (ICC) for ghrelin and in situ hybridization (ISH) for ghrelin mRNA. Ghrelin was expressed in especially the corpus but also the antrum of the stomach of all groups studied. Cells positive for ghrelin and ghrelin mRNA in the stomach were reduced in high-fat fed mice. In contrast, ghrelin expression was not affected by fasting. The reduction in ghrelin expression in the high-fat fed mice was associated with a reduction in plasma levels of ghrelin, whereas after fasting, when expression rate was not altered, there was an increase in plasma ghrelin. In conclusion, ghrelin is highly expressed in the corpus and antrum of the stomach of C57BL/6J mice. This expression is reduced in obesity, whereas fasting has no effect.