Pharmacokinetics of the Urokinase Receptor-Derived Peptide UPARANT After Single and Multiple Doses Administration in Rats.

BACKGROUND AND OBJECTIVES: UPARANT has emerged as a novel therapeutic agent with the potential to treat ocular diseases as assessed by studies in animal models. Since limited information is available on the pharmacokinetics of UPARANT, the aim of this study is to evaluate its pharmacokinetics after single and multiple ascending dose (SAD and MAD) administration in rats. METHODS: Male (n = 27) and female (n = 27) Sprague-Dawley rats were divided into six groups (n = 9/sex/group). UPARANT was administered via subcutaneous injection as single (10, 50 or 100 mg/kg; day 1) and multiple (10, 50 or 100 mg/kg/day; 7 consecutive days; day 7) dosing. Blood samples were collected on day 1 (pre-dose, 0.5, 1, 2, 4, 8 and 24 h post dose) and day 7 (pre-dose, 0.5, 1, 2, 4, 8, 24, 48 and 192 h post dose). The plasma concentration of UPARANT was determined by a validated liquid chromatography mass spectrometry method. RESULTS: The plasma concentration-time profiles of UPARANT were similar in SAD and MAD administration in both male and female rats. The compound reached maximum plasma concentration (Cmax) at 1-2 h with a slow apparent plasma clearance and a moderate apparent volume of distribution. Moreover, SAD administration revealed a non-proportional increase in Cmax and in the area under the plasma concentration-time curve (AUCinf), whereas a dose-proportional increase in AUCinf was shown after MAD administration. Regarding the extent of accumulation, the data suggest negligible accumulation of the compound after multiple administrations. CONCLUSION: The pharmacokinetics of UPARANT were not sex-related, and there was negligible accumulation in plasma after 7 days of treatment. However, the compound exhibited no dose-proportional pharmacokinetics after single and multiple ascending subcutaneous dosing.

Antitumor Effect of the Atypical Retinoid ST1926 in Acute Myeloid Leukemia and Nanoparticle Formulation Prolongs Lifespan and Reduces Tumor Burden of Xenograft Mice.

Acute myeloid leukemia (AML) is one of the most frequent types of blood malignancies. It is a complex disorder of undifferentiated hematopoietic progenitor cells. The majority of patients generally respond to intensive therapy. Nevertheless, relapse is the major cause of death in AML, warranting the need for novel treatment strategies. Retinoids have demonstrated potent differentiation and growth regulatory effects in normal, transformed, and hematopoietic progenitor cells. All-trans retinoic acid (ATRA) is the paradigm of treatment in acute promyelocytic leukemia, an AML subtype. The majority of AML subtypes are, however, resistant to ATRA. Multiple synthetic retinoids such as ST1926 recently emerged as potent anticancer agents to overcome such resistance. Despite its lack of toxicity, ST1926 clinical development was restricted due to its limited bioavailability and rapid excretion. Here, we investigate the preclinical efficacy of ST1926 and polymer-stabilized ST1926 nanoparticles (ST1926-NP) in AML models. We show that sub-µmol/L concentrations of ST1926 potently and selectively inhibited the growth of ATRA-resistant AML cell lines and primary blasts. ST1926 induced-growth arrest was due to early DNA damage and massive apoptosis in AML cells. To enhance the drug's bioavailability, ST1926-NP were developed using Flash NanoPrecipitation, and displayed comparable anti-growth activities to the naked drug in AML cells. In a murine AML xenograft model, ST1926 and ST1926-NP significantly prolonged survival and reduced tumor burden. Strikingly, in vivo ST1926-NP antitumor effects were achieved at four fold lower concentrations than the naked drug. These results highlight the promising use of ST1926 in AML therapy and encourage its further development. Mol Cancer Ther; 16(10); 2047-57. ©2017 AACR.

The Urokinase Receptor-Derived Peptide UPARANT Recovers Dysfunctional Electroretinogram and Blood-Retinal Barrier Leakage in a Rat Model of Diabetes.

Purpose: The activation of the urokinase-type plasminogen activator and its receptor system is associated with retinal diseases. Among peptide inhibitors of this system, UPARANT acts by preventing the onset of pathologic signs of neovascular ocular diseases. We investigated whether systemic UPARANT may act in a therapeutic regimen by suppressing the retinal damage that characterizes diabetic retinopathy using a rat model of streptozotocin-induced diabetes. Methods: In healthy rats, plasma, eye, and retina concentrations of UPARANT were evaluated by mass spectrometry. In rat models of streptozotocin-induced diabetes, the appearance of diabetic retinopathy was assessed by electroretinogram (ERG). UPARANT was then administered at different dosages and daily regimens. ERG recording, Evans blue perfusion, and real-time PCR were used to evaluate UPARANT efficacy. UPARANT safety was also determined. Results: UPARANT was found in plasma, eye, and retina soon after its administration and remained detectable after 24 hours. Between the 4th and the 5th week after diabetes onset, UPARANT at 8 mg/kg (daily for 5 days) was effective in recovering dysfunctional ERG. Three-day treatments at 8 mg/kg or a half dose for 5 days were ineffective. ERG recovery lasted approximately 2 weeks. ERG recovery was accompanied by restored blood-retinal barrier integrity and inhibition of inflammatory and angiogenic responses. UPARANT showed a safety profile. Conclusions: These data suggest that targeting the urokinase-type plasminogen activator and its receptor system by systemic UPARANT is a potential therapeutic approach for the treatment of early diabetic retinopathy, thus providing a potential alternative approach to delay disease progression in humans.

The synthetic retinoid ST1926 as a novel therapeutic agent in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most frequent soft tissue sarcoma in children. Despite multiple attempts at intensifying chemotherapeutic approaches to treatment, only moderate improvements in survival have been made for patients with advanced disease. Retinoic acid is a differentiation agent that has shown some antitumor efficacy in RMS cells in vitro; however, the effects are of low magnitude. E-3-(4'-hydroxyl-3'-adamantylbiphenyl-4-yl) acrylic acid (ST1926) is a novel orally available synthetic atypical retinoid, shown to have more potent activity than retinoic acid in several types of cancer cells. We used in vitro and in vivo models of RMS to explore the efficacy of ST1926 as a possible therapeutic agent in this sarcoma. We found that ST1926 reduced RMS cell viability in all tested alveolar (ARMS) and embryonal (ERMS) RMS cell lines, at readily achievable micromolar concentrations in mice. ST1926 induced an early DNA damage response (DDR), which led to increase in apoptosis, in addition to S-phase cell cycle arrest and a reduction in protein levels of the cell cycle kinase CDK1. Effects were irrespective of TP53 mutational status. Interestingly, in ARMS cells, ST1926 treatment decreased PAX3-FOXO1 fusion oncoprotein levels, and this suppression occurred at a post-transcriptional level. In vivo, ST1926 was effective in inhibiting growth of ARMS and ERMS xenografts, and induced a prominent DDR. We conclude that ST1926 has preclinical efficacy against RMS, and should be further developed in this disease in clinical trials.

Pharmacokinetic Profile of µSMIN Plus™, a new Micronized Diosmin Formulation, after Oral Administration in Rats.

Diosmin is a naturally occurring flavonoid present in citrus fruits and other plants belonging to the Rutaceae family. It is used for the treatment of chronic venous insufficiency (CVI) for its pheblotonic and vaso-active properties, safety and tolerability as well. The aim of the current in vivo study was to investigate the pharmacokinetic profile of a branded micronized diosmin (µSMIN Plus™) compared with plain micronized diosmin in male Sprague-Dawley rats. After oral administration by gastric gavage, blood samples were collected via jugular vein catheters at regular time intervals from baseline up to 24 hours. Plasma concentrations were assessed by LC/MS. For each animal, the following pharmacokinetic parameters were calculated using a non-compartmental analysis: maximum plasma drug concentration (Cmax), time to reach Cmax (Tmax), area under the plasma concentration-time curve (AUC0-last), elimination half-life (t½), and relative oral bioavailability (%F). The results of the current study clearly showed an improvement in the pharmacokinetic parameters in animals treated with µSMIN Plus™ compared with animals treated with micronized diosmin. In particular, µSMIN Plus™ showed a 4-fold increased bioavailability compared with micronized diosmin. In conclusion, the results from the current study provided a preliminary pharmacokinetic profile for µSMIN Plus™, which may represent a new tool for CVI management.

Optimization of a rapid capillary electrophoresis ESI-IT tandem mass spectrometry method for the analysis of short-chain carnitines in human plasma.

A capillary electrophoresis (CE) method coupled to electrospray ionization ion trap tandem mass spectrometry (ESI-IT-MS/MS) is described for the rapid analysis of carnitine, acetylcarnitine, and propionylcarnitine in human plasma. Optimization of the procedure was achieved by a reduced sample pretreatment and after examining several physicochemical parameters that influence both the CE separation and the MS analytes detection. The analysis of total carnitine in human plasma after hydrolysis of short-chain metabolites is also shown. The analysis of carnitine and metabolites was obtained in less than 10 min using a 200 mM ammonium formate buffer, pH 2.5, with high sensitivity and specificity using the MS detection in product ion scan mode. The method was tested for quantitative recovery using dialyzed human plasma as matrix and showed linearity in the concentrations ranges 20-160, 1-32, and 0.25-8 microM for carnitine, acetylcarnitine, and propionylcarnitine with (squared) correlation coefficients of 0.9984, 0.9995, and 0.9991, respectively. The intraday and intermediate analysis repeatability and accuracy are within 15% of relative standard deviation (RSD) at low, medium, and high concentration and within/or slight exceeding 20% at the lower limit of quantitation (LLOQ). The method is sensitive for determining carnitine and its metabolites in human plasma with high specificity.

Urinary excretion of L-carnitine and its short-chain acetyl-L-carnitine in patients undergoing carboplatin treatment.

PURPOSE: To evaluate the effect of the anti-cancer drug carboplatin on plasma concentrations and urinary excretion of L-carnitine (LC) and its main ester, acetyl-L-carnitine (ALC), in cancer patients. METHODS: Plasma and urine concentrations of LC and ALC from 11 patients on carboplatin therapy (1 h intravenous infusion; AUC dose 4.8 +/- 1.1 mg/ml min) in combination with docetaxel, paclitaxel or vinorelbine, were determined by high-performance liquid chromatography with fluorimetric detection. RESULTS: Before carboplatin therapy, the mean +/- SD plasma concentrations of LC and ALC were 47.8 +/- 10.9 and 7.04 +/- 1.04 nmoles/ml, respectively, and remained constant throughout the entire study period. In contrast, urinary excretion of LC and ALC, increased significantly during the chemotherapy from 115 +/- 105 to 480 +/- 348 micromoles/day (P < 0.01) and from 41 +/- 41 to 89 +/- 52 micromoles/day (P < 0.05) for LC and ALC, respectively, subsequently reverting to normal 6 days after the end of chemotherapy. Similarly, the renal clearance of LC and ALC increased substantially during chemotherapy from 1.67 +/- 1.43 to 9.05 +/- 9.52 ml/min (P < 0.05) and from 4.02 +/- 4.51 to 7.97 +/- 5.05 ml/min (P = not significant) for LC and ALC, respectively, reverting to normal 6 days after the end of chemotherapy. Plasma concentrations and urinary excretion of glucose, phosphate and urea nitrogen and creatinine clearance, however, were not affected by carboplatin therapy, indicating no impaired kidney function. CONCLUSION: Treatment with carboplatin was associated with a marked urinary loss of LC and ALC, most likely due to inhibition of carnitine reabsorption in the kidney.

Rapid determination of short chain carnitines in human plasma by electrospray ionisation-ion trap mass spectrometry using capillary electrophoresis instrument as sampler.

A capillary electrophoresis apparatus was used as sampler for flow injection analysis (FIA) in tandem mass spectrometry of L-carnitine and its acetyl- and propionyl-metabolites in human plasma. The capillary electrophoresis instrument was coupled to the ion trap mass spectrometer by an electrospray ionization coaxial sheath liquid interface. The electrophoresis capillary introduced the sample directly into the source by applying a prolonged sample injection. The use of the capillary electrophoresis apparatus miniaturised the FIA procedure, substantially reducing the quantities of solvents and samples used, and allowed rapid automated sequential analyses. The method was optimised and validated using a dialyzed human plasma matrix. The plasma samples were analysed after a simple, rapid deproteinisation procedure with acetonitrile and diluted 70 times before direct injection into the mass spectrometer for product ion scan MS/MS analysis in positive ionisation. The total analysis time was 5 min, including capillary preconditioning and acquisition time (3 min). The method was sensitive, allowing the determination of L-, L-acetyl- and L-propionyl-carnitines at 140, 14 and 3.6 nM concentrations (injected values) corresponding to lower limit of quantitation values in plasma of 10, 1 and 0.25 microM, respectively. The method was processed for full validation and applied to the analysis of L-carnitine and its short chain derivatives in human plasma samples.

Uptake of L-carnitine and its short-chain ester propionyl-L-carnitine in the isolated perfused rat liver.

Hepatic uptake of propionyl-L-carnitine (PLC) and L-carnitine (LC) was assessed with the impulse-response technique in the single-pass perfused rat liver. The experiments involved a rapid injection (impulse) of a mixture of the radiolabeled test compound (PLC or LC) and a reference compound (sucrose) into portal vein inflow and collection and radiochemical analysis (response) of the venous outflowing perfusate samples. The impulse injection was made in the presence of increasing unlabeled background concentrations of PLC (0-50 microM) or LC (50-500 microM) perfusing the liver. The hepatic uptake was minimal or negligible for LC, whereas the hepatic influx clearance was found to be low (0.095 ml/s equivalent to 5.7 ml/min) for PLC relative to the perfusate flow rate (30 ml/min). When background concentrations of PLC were increased (from 1-50 microM), the influx clearance was reduced in a concentration-dependent behavior, indicating partial saturation of the entry of compound into hepatocytes. PLC was taken up into hepatocytes via a unidirectional transport process with negligible efflux. The hepatic uptake of PLC was significantly reduced in the presence of unlabeled LC (500 microM), indicating an inhibition of the sinusoidal membrane transport of PLC by LC. The study showed the sinusoidal membrane is a permeability barrier to the entry of PLC and LC into hepatocytes, and it is the site of a common carrier-mediated transporter for both compounds.

Carnitine replacement in end-stage renal disease and hemodialysis.

In patients with chronic renal failure, not yet undergoing hemodialysis (HD), plasma acylcarnitines accumulate in part due to a decreased renal clearance of esterified carnitine moieties. In these patients, a high acylcarnitine/free-carnitine ratio is usually found in plasma. Patients undergoing maintenance HD, usually present with plasma carnitine insufficiency, due to accumulation of metabolic intermediates combined with impaired carnitine biosynthesis, reduced protein intake and increased removal via HD. Plasma carnitine concentrations rapidly decrease to 40% of baseline level during the dialysis session, with a slow restoration of the carnitine concentration during the interdialytic period, mainly from organs of storage (skeletal muscle). Dietary intake also plays an important role in carnitine homeostasis of HD patients since the prevalence of malnutrition ranges from 18% to 75% of these cases. This could differentially affect various body compartments, with clinical consequences such as impaired muscle function, decreased wound healing, altered ventilatory response, and abnormal immune function. Repeated hemodialytic treatments are associated with decreased carnitine stores in skeletal muscle. The administration of intravenous L-carnitine (LC) postdialysis replenishes the free carnitine removed from the blood and contributes to replenishment of muscle carnitine content. LC supplementation in selected uremic patients may yield clinical benefits by ameliorating several conditions, such as erythropoietin-resistant anemia, decreased cardiac performance, intradialytic hypotension, muscle symptoms, as well as impaired exercise and functional capacities. Furthermore, LC may positively influence the nutritional status of HD patients by promoting a positive protein balance, and by reducing insulin resistance and chronic inflammation, possibly through an effect on leptin resistance.