Impact of Precipitation of Antibody Therapeutics After Subcutaneous Injection on Pharmacokinetics and Immunogenicity.

Antibody therapeutics with poor solubility in the subcutaneous matrix may carry unintended risks when administered to patients. The objective of this work was to estimate the risk of antibodies that precipitate in vitro at neutral pH by determining the impact of poor solubility on distribution of the drug from the injection site as well as immunogenicity in vivo. Using fluorescence imaging in a mouse model, we show that one such precipitation-prone antibody is retained at the injection site in the subcutaneous space longer than a control antibody. In addition, we demonstrate that retention at the injection site through aggregation is concentration-dependent and leads to macrophage association and germinal center localization. Although there was delayed disposition of the aggregated antibody to draining lymph nodes, no overall impact on the immune response in lymph nodes, systemic exposure of the antibody, or enhancement of the anti-drug antibody response was evident. Unexpectedly, retention of the precipitated antibody in the subcutaneous space delayed the onset of the immune response and led to an immune suppressive response. Thus, we conclude that precipitation due to poor solubility of high doses of antibody formulations delivered subcutaneously may not be of special concern in terms of exposure or immunogenicity.

A multi-center preclinical study of gadoxetate DCE-MRI in rats as a biomarker of drug induced inhibition of liver transporter function.

Drug-induced liver injury (DILI) is a leading cause of acute liver failure and transplantation. DILI can be the result of impaired hepatobiliary transporters, with altered bile formation, flow, and subsequent cholestasis. We used gadoxetate dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), combined with pharmacokinetic modelling, to measure hepatobiliary transporter function in vivo in rats. The sensitivity and robustness of the method was tested by evaluating the effect of a clinical dose of the antibiotic rifampicin in four different preclinical imaging centers. The mean gadoxetate uptake rate constant for the vehicle groups at all centers was 39.3 +/- 3.4 s-1 (n = 23) and 11.7 +/- 1.3 s-1 (n = 20) for the rifampicin groups. The mean gadoxetate efflux rate constant for the vehicle groups was 1.53 +/- 0.08 s-1 (n = 23) and for the rifampicin treated groups was 0.94 +/- 0.08 s-1 (n = 20). Both the uptake and excretion transporters of gadoxetate were statistically significantly inhibited by the clinical dose of rifampicin at all centers and the size of this treatment group effect was consistent across the centers. Gadoxetate is a clinically approved MRI contrast agent, so this method is readily transferable to the clinic. CONCLUSION: Rate constants of gadoxetate uptake and excretion are sensitive and robust biomarkers to detect early changes in hepatobiliary transporter function in vivo in rats prior to established biomarkers of liver toxicity.

Response-Derived Input Function Estimation for Dynamic Contrast-Enhanced MRI Demonstrated by Anti-DLL4 Treatment in a Murine U87 Xenograft Model.

PURPOSE: Dynamic contrast-enhanced magnetic resonance imaging (DCE MRI) is an accepted method to evaluate tumor perfusion and permeability and anti-vascular cancer therapies. However, there is no consensus on the vascular input function estimation method, which is critical to kinetic modeling and K trans estimation. This work proposes a response-derived input function (RDIF) estimated from the response of the tumor, modeled as a linear, time-invariant (LTI) system. PROCEDURES: In an LTI system, an unknown input can be estimated from the system response. If applied to DCE MRI, this method would eliminate need of distal image-derived inputs, model inputs, or reference regions. The RDIF method first determines each tumor pixel's best-fit input function, and then combines the individual fits into a single input function for the entire tumor. The method was tested with simulations and a xenograft study with anti-vascular drug treatment. RESULTS: Simulations showed successful estimation of input function expected values and good performance in the presence of noise. In vivo, significant reductions in K trans and AUC occurred 2 days following anti-delta-like ligand 4 treatment. The in vivo study results yielded K trans consistent with published data in xenograft models. CONCLUSION: The RDIF method for DCE analysis offers an alternative, easy-to-implement method for estimating the input function in tumors. The method assumes that during the DCE experiment, the changes observed by MRI result solely from vascular perfusion and permeability kinetics, and that information can be used to model the input function. Importantly, the method is demonstrated in a murine xenograft study to yield K trans results consistent with literature values and suitable for compound studies.

Hepatic glycogen cycling contributes to glucose lowering effects of the glucokinase activator LCZ960.

Glucokinase (GK) acts as a glucose sensor by facilitating glucose phosphorylation into glucose-6-phosphate (G6P) in the liver and pancreas, the two key target tissues. LCZ960, a glucokinase activator exerts a stimulatory effect on GK activity in hepatocytes in vitro. This study aimed to verify in vivo that LCZ960 stimulates glucose uptake primarily through a mechanism involving hepatic GK activation. Acute and chronic LCZ960 treatment-induced changes in glycemia and hepatic glucose turnover were measured in high fat diet-induced obese (DIO) mice and rats. G6P production and glycogen cycling were quantified by (13)C-MR spectroscopy during a [1-(13)C]glucose infusion, followed by a pulse-chase with [(12)C]glucose to mimic postprandial conditions in rats. Acute treatment with LCZ960 dose-dependently reduced blood glucose without causing hypoglycemia in DIO mice. Chronic LCZ960 treatment maintained normoglycemia and improved glucose tolerance without increased insulin secretion in DIO mice and rats. In rats, LCZ960 stimulated a 240% increase (P<0.05) in the glycogen synthase flux. However, due to a much higher glycogen breakdown (LCZ960: 48 ± 15 vs control: 4 ± 1µmol/kg/min, P<0.05), this translated to only a 46% (ns) increase in glycogen storage (Vsyn net, LCZ960: 64±26 vs control: 43 ± 6 µmol/kg/min). Despite a 4-fold increase in hepatic glycogen turnover (LCZ960: 36.0 ± 5.5% vs control: 8.3 ± 2.0%), LCZ960 did not impact glucose-stimulated G6P accumulation. LCZ960 did not cause hypoglycemia in DIO rodents. Under hyperglycemic conditions, LCZ960 induced a robust increase in hepatic glycogen cycling. Since net hepatic glycogen storage is diminished in type 2 diabetes patients, stimulation of glycogen synthesis may contribute to the anti-hyperglycemic properties of glucokinase activation.

Insulin-stimulated mitochondrial adenosine triphosphate synthesis is blunted in skeletal muscles of high-fat-fed rats.

Physiologic elevation of insulin levels induces a significant increase in muscle adenosine triphosphate (ATP) synthesis rate in normal individuals, indicative of an appropriate acceleration in mitochondrial activity. However, the stimulatory effect of insulin is diminished in insulin-resistant patients. In the absence of similar data from preclinical models, the present study investigated the inhibitory effects of increased dietary fat intake on insulin-stimulated ATP synthesis rates in rats. After being placed on a high-fat diet for 8 weeks (n = 10), diet-induced obese male Sprague-Dawley rats were tested against age-matched control rats (n = 9) on a normal chow diet. Muscle ATP synthase flux rates were measured under anesthesia by in vivo (31)P saturation transfer both before and during a euglycemic-hyperinsulinemic clamp. The glucose infusion rates observed during the clamp revealed impaired peripheral insulin sensitivity in the high-fat-fed rats when compared with the age-matched control rats. Under baseline conditions (ie, low insulin), the muscle ATP synthesis rates of high-fat-fed rats were approximately 30% lower (P < .05) than those in chow-fed rats. Moreover, chow-fed animals showed a significant increase (25%, P < .05 vs basal) in muscle ATP synthesis activity upon insulin stimulation, whereas high-fat-fed animals displayed no substantial change. These data demonstrated for the first time in a preclinical model that the insulin challenge not only facilitates an improvement in the dynamic range of ATP turnover measurement by (31)P saturation transfer between normal and insulin-resistant rats, but also mimics challenge that is relevant for pharmacologic studies on antidiabetic drugs aimed at improving mitochondrial function.

Diet-induced modulation of mitochondrial activity in rat muscle.

Growing evidence supports the theory that mitochondrial dysfunction is an underlying cause of intramyocellular lipid (IMCL) accumulation and insulin resistance. Here, we hypothesized that high dietary fat (HF) intake could trigger changes in mitochondrial activity such that fatty acid oxidation is impaired in muscle and contributes to an elevation in intramyocellular lipid (IMCL) levels. Muscle mitochondrial activity was determined in vivo through measurement of the F(1)F(0) ATP synthase flux, the terminal step in the oxidative phosphorylation process. An initial study comparing rats on normal chow diet with rats on an HF diet revealed strong correlations between muscle ATP synthesis rates, IMCL levels and whole body glucose tolerance. Results obtained from two latter studies showed multiphasic responses to dietary intervention. Initially, the ATP synthesis rates decreased as much as 50% within 24 h of raising the fat content in the diet to 60% of the caloric intake. These rates eventually returned to normal values after 2-3 wk on the HF regimen, seemingly to prevent further IMCL accumulation. Only beyond 1 mo on the HF diet did results consistently show ATP synthesis rates to diminish by 30-50% accompanied by steadily augmenting IMCL levels. Interestingly, switching back to a chow diet after 3 wk of HF feeding reversed the initial diet-induced changes. Although the muscle mitochondrial system may initially offer enough compliance to counteract lipid surplus, these in vivo data suggest a vicious long-term cycle among mitochondrial dysfunction, IMCL accumulation, and glucose intolerance in the rat.