Performance of 99mTc-aprotinin scintigraphy for diagnosing light chain (AL) cardiac amyloidosis confirmed by endomyocardial biopsy.

BACKGROUND: Light chain (AL) cardiac amyloidosis is associated with a poor prognosis. Diagnosing at an early stage is critical for treatment and the management of cardiac complication. PURPOSE: We aimed to evaluate the diagnostic performance of 99mTc-aprotinin images in patients with AL cardiac amyloidosis. METHODS AND RESULTS: 99mTc-aprotinin scintigraphy and endomyocardial biopsy were performed in 10 patients with suspected amyloidosis. Endomyocardial biopsy showed amyloid deposits in 5 of 10 patients. 99mTc-aprotinin (planer image) was positive in 4 of 5 patients who had amyloid deposits in endomyocardial biopsy. On the other hand, all 5 patients without amyloid deposits were negative in planer image. 99mTc-aprotinin (SPECT/CT image) was positive in all 5 patients who had amyloid deposits. CONCLUSIONS: 99mTc-aprotinin scintigraphy is valuable for the non-invasive diagnosis of AL cardiac amyloidosis.

Formulation Optimization of Human Insulin Loaded Microspheres for Controlled Oral Delivery Using Response Surface Methodology.

OBJECTIVES: The objectives of the present investigation were to prepare recombinant human insulin entrapped Eudragit-S100 microspheres containing protease inhibitors and to precisely analyze the outcome of different formulation variables on the microspheres properties using a response surface methodology to develop an optimized formulation with desirable features. METHODS: A central composite design was employed to produce microspheres of therapeutic protein by w/o/w multiple emulsion solvent evaporation technique using Eudragit S-100 as coating material and polyvinyl alcohol as a stabilizer. The effect of formulation variables (independent variables) that is levels of Eudragit S-100 (X1), therapeutic protein (X2), volumes of inner aqueous phase (X3) and external aqueous phase (X4) on dependant variables, that are encapsulation efficiency (Y1), drug release at pH 1.2 after 2 h (Y2) and drug release at pH 7.4 after of 2 h (Y3) were evaluated. RESULTS: The significant terms in the mathematical models were generated for each response parameter using multiple linear regression analysis and analysis of variance. All the formulation variables except the volume of external aqueous phase (X4) exerted a significant effect (P <0.05) on drug encapsulation efficiency (Y1) whereas first two variables, namely the levels of Eudragit S-100 (X1) and therapeutic protein (X2) materialized as the determining factors which significantly influenced drug release at pH 1.2 after 2 h (Y2) and drug release at pH 7.4 after of 2 h (Y3). The formulation was numerically optimized by framing the constraints on the dependent and independent variables using the desirability approach. The experimental values for Y1 and Y2 of optimized formulation were found to be 77.65% and 3.64%, respectively which were quite closer to results suggested by software. CONCLUSION: The results recorded indicate that the recombinant human insulin loaded Eudragit S-100 microspheres containing aprotinin have the benefits of higher loading efficiency, pH responsive and prolonged release characteristics, which may help to carry insulin to the optimum site of absorption.

Aprotinin revisited: formulation, characterization, biodistribution and therapeutic potential of new aprotinin microemulsion in acute pancreatitis.

The aim of this study was to develop aprotinin-loaded microemulsion (MA) for intravenous administration and evaluate the biodistribution and therapeutic potential of developed formulation in acute pancreatitis models in rats. Phase diagrams were constructed to identify microemulsion region and the optimal microemulsion was evaluated for physicochemical properties and treatment effect in rats, and comparisons made with the solution of aprotinin (SA). To evaluate the biodistribution of the drug by gamma scintigraphy aprotinin was radiolabeled with (99m)Tc radionuclide. Mild and severe acute pancreatitis was induced in rats by subcutaneous injections of cerulein and introductal infusion of 3% sodium taurocholate into the bile-pancreatic duct, respectively. In addition, serum amylase and pancreatic tissue myeloperoxidase activities were measured to evaluate the pancreatic damage. According to gamma scintigraphy and biodistribution studies, accumulation times and distribution of (99m)Tc-MA and SA were different. While MA was highly uptake by reticuloendothelial system, SA was mostly excreted by kidneys and bladder. Compared with the mild acute pancreatitis group, treatment with MA significantly decreased the serum amylase activity and pancreas myeloperoxidase activity. Furthermore, the protease inhibitor molecule aprotinin has therapeutic potential in acute pancreatitis. Finally, MA may be suggested as a promising alternative for treatment of acute pancreatitis.

Fibrin chain cross-linking, fibrinolysis, and in vivo sealing efficacy of differently structured fibrin sealants.

In this study, we compared the sealing characteristics and efficacy of a fibrin sealant with reduced plasminogen (FS-rplg) and a fibrin sealant with aprotinin as a fibrinolysis inhibitor (FS-apr). The relevant sealing characteristics including clot structure, fibrin chain cross-linking, and clot lysis were tested in the laboratory. The sealing efficacy was then investigated in a follow-up animal model to determine differences in the in vivo sealing properties. A total of 46 animals were available for the final analysis with 23 animals in each treatment arm. In conclusion, we saw differences in vitro between FS-rplg and FS-apr in ultrastructure and α-chain cross-linking rates as well as in the rate of fibrinolysis. These differences may explain the significantly enhanced sealing efficacy in FS-apr compared to FS-rplg shown in vivo in a rabbit intestinal model.

Evaluation of microemulsion and liposomes as carriers for oral delivery of transforming growth factor alpha in rats.

The aim of the present study is to develop microemulsion and liposome carrier systems for oral administration of transforming growth factor alpha (TGF-α) and to investigate the effects of these carrier systems on the gastrointestinal efficiency in rats. Microemulsion (w/o) and liposomes (MLV) were developed and characterised. The carrier systems were administered intragastrically by gastric cannula to male Wistar rats. The highest reduction in the basal acid secretion was observed in the microemulsion containing TGF-α and aprotinin group (TAME).The gastric mucus secretion in microemulsion containing TGF-α (TME) and TAME treatment groups increased significantly compared to the other groups. TGF-α levels in both stomach and duodenum were significantly increased in the TAME group. As a result, it was determined through confocal laser scanning microscope (CLSM) studies that exogenous-applied TGF-α attached to endogenous EGF receptors. The microemulsion formulation was found to be a more suitable carrier system for oral administration of TGF-α.

Increasing stability and toxicity of Pseudomonas exotoxin by attaching an antiproteasic Peptide.

Trypsin-like activities are present within the endocytic pathway and allow cells to inactivate a fraction of incoming toxins, such as Pseudomonas exotoxin (PE), that require endocytic uptake before reaching the cytosol to inactivate protein synthesis. PE is a favorite toxin for building immunotoxins. The latter are promising molecules to fight cancer or transplant rejection, and producing more active toxins is a key challenge. More broadly, increasing protein stability is a potentially useful approach to improve the efficiency of therapeutic proteins. We report here that fusing an antiproteasic peptide (bovine pancreatic trypsin inhibitor, BPTI) to PE increases its toxicity to human cancer cell lines by 20-40-fold. Confocal microscopic examination of toxin endocytosis, digestion, and immunoprecipitation experiments showed that the fused antiproteasic peptide specifically protects PE from trypsin-like activities. Hence, the attached BPTI acts as a bodyguard for the toxin within the endocytic pathway. Moreover, it increased the PE elimination half-time in mice by 70%, indicating that the fused BPTI stabilizes the toxin in vivo. This BPTI-fusion approach may be useful for protecting other circulating or internalized proteins of therapeutic interest from premature degradation.

Investigation of a potential scintigraphic tracer for imaging apoptosis: radioiodinated annexin V-Kunitz protease inhibitor fusion protein.

Radiolabeled annexin V (ANV) has been widely used for imaging cell apoptosis. Recently, a novel ANV-Kunitz-type protease inhibitor fusion protein, ANV-6L15, was found to be a promising probe for improved apoptosis detection based on its higher affinity to phosphatidylserine (PS) compared to native ANV. The present paper investigates the feasibility of apoptosis detection using radioiodinated ANV-6L15. Native ANV and ANV-6L15 were labeled with iodine-123 and iodine-125 using Iodogen method. The binding between the radioiodinated proteins and erythrocyte ghosts or chemical-induced apoptotic cells was examined. ANV-6L15 can be radioiodinated with high yield (40%-60%) and excellent radiochemical purity (>95%). (123)I-ANV-6L15 exhibited a higher binding ratio to erythrocyte ghosts and apoptotic cells compared to (123)I-ANV. The biodistribution of (123)I-ANV-6L15 in mice was also characterized. (123)I-ANV-6L15 was rapidly cleared from the blood. High uptake in the liver and the kidneys may limit the evaluation of apoptosis in abdominal regions. Our data suggest that radiolabeled ANV-6L15 may be a better scintigraphic tracer than native ANV for apoptosis detection.

Population pharmacokinetic analysis and dosing regimen optimization of aprotinin in neonates and young infants undergoing cardiopulmonary bypass.

The objective of this study was to determine an optimal dosing regimen for maintaining the therapeutic target range of aprotinin in neonates and young infants during cardiopulmonary bypass (CPB). A total of 27 patients scheduled for open heart surgery were enrolled. Aprotinin was administered a 25 000 KIU (kallikrein inhibition unit)/kg bolus before operation, a 35 000 KIU/kg for CPB circuit priming, and a 12 500 KIU/kg/hour continuous infusion intra- and immediate postoperative period. Blood samples were obtained at 12 time points per patient. Population pharmacokinetic modeling and Monte-Carlo simulations were used to optimize the aprotinin dosing regimen. No mortality or aprotinin-related complication was encountered. A CPB adjusted 2-compartment model best fit the data. Clearance was 687 mL/hour during CPB and 350 mL/hour pre- and post-CPB, and corresponding volumes of distribution were 1577 mL and 1352 mL, respectively. The simulations conducted showed that more than twice the dose administered in this study is required to maintain the target concentration of aprotinin. The pharmacokinetics of aprotinin appears to be affected more sensitively by CPB in neonates and young infants than in adults. Therefore, dosage adjustment considering these pharmacokinetic differences and the influence of CPB is needed in neonates and young infants.

Aprotinin and renal dysfunction.

Aprotinin is a polypeptide serine protease inhibitor used to prevent bleeding and need for transfusions in patients having heart surgery. A recent analysis of an observational study data set suggested the use of aprotinin was associated with an increased risk of developing renal failure. The present article reviews the data from basic science studies in tissues, animals and man together with the data from observational studies and randomised controlled trials. The interpretation of the data is hampered owing to the use of different endpoints to describe mild/moderate renal impairment. Nonetheless, the evidence points to aprotinin use being associated with a transient small rise in plasma creatinine concentration in certain patients. There is no evidence for an increased risk of developing new renal failure requiring dialysis/renal replacement therapy.

Preliminary experience of 99mTc-Aprotinin scintigraphy in amyloidosis.

BACKGROUND AND AIM: Radio-labelled Aprotinin has been shown to bind with amyloid fibrils in vitro as well as in vivo. The aim was to test the usefulness of 99mTc-Aprotinin imaging in systemic amyloidosis. METHODS: Thirty-five cases who had 99mTc-Aprotinin scans for the assessment of systemic amyloidosis were reviewed retrospectively. Eighteen had biopsy-proven amyloidosis and 17 were controls (amyloidosis was excluded by negative biopsies and non-invasive tests). Five of 18 patients with amyloidosis had final diagnosis of cardiac amyloid. RESULTS: Physiological uptake of 99mTc-Aprotinin was noted in the urinary tract (kidneys and bladder) and in the liver of all patients and controls; and non-specific uptake of 99mTc-Aprotinin was visualised in the spleen and oro-facial structures in the majority of both groups. Myocardial 99mTc-Aprotinin uptake was noted in all five patients with final diagnosis of cardiac amyloidosis and in none of the 30 subjects who did not have cardiac amyloid. The median heart to background uptake ratio was 2.0 in cardiac amyloid patients and 1.1 in subjects without cardiac amyloid (P = 0.0004). Single Photon Emission Tomography (SPECT) studies of the thorax confirmed that the site of uptake lay within the myocardium. In the amyloidosis group, site-specific 99mTc-Aprotinin uptake was also identified in the subcutaneous tissue of the legs and in a breast nodule shown to be positive for amyloidosis on biopsy. CONCLUSIONS: 99mTc-Aprotinin imaging may be a useful non-invasive method for the assessment of the presence and extent of extra-abdominal amyloid, particularly cardiac amyloidosis. It has little role in diagnosis of amyloidosis involving the oro-facial and abdominal structures.

Design and evaluation of a chitosan-aprotinin conjugate for the peroral delivery of therapeutic peptides and proteins susceptible to enzymatic degradation.

One main barrier for the peroral administration of therapeutic peptides and proteins is the enzymatic barrier, that is mediated by luminally secreted and membrane bound proteolytic enzymes. It was the aim of the study to synthesise, characterise and evaluate a novel polymer-inhibitor conjugate in order to improve the bioavailability of orally-administered peptides and proteins. The trypsin/chymotrypsin inhibitor aprotinin was covalently bound to chitosan. The percentage of the inhibitor in the polymer-inhibitor conjugate (m/m) was determined to be between 1.11 +/- 0.36 and 1.92 +/- 0.05%. In vitro enzyme assays clearly demonstrated the potential of the novel conjugate to inhibit trypsin and chymotrypsin. Moreover, studies in rats were performed to evaluate the efficacy of the conjugate in vivo. Eight hours after oral administration of tablets containing insulin and the novel chitosan-aprotinin conjugate, the mean blood glucose level decreased to 84 +/- 6%. In contrast, the mean blood glucose level in the control group increased to 121 +/- 8% of the initial measured blood glucose level. In conclusion it was demonstrated that chitosan-aprotinin conjugate represents a novel and promising tool for the oral administration of therapeutic peptides and proteins susceptible to enzymatic degradation caused by trypsin and chymotrypsin.

Localization of tubular uptake segment of filtered Cystatin C and Aprotinin in the rat kidney.

AIM: The renal tubular uptake of 125I-Aprotinin (*Ap) is on average located more superficially than its filtration site, causing transfer of some of *Ap filtered in deep to more superficial cortical zones. 125I-Cystatin C (*Cy) showed less uptake in deep cortical zones than Ap, suggesting a longer and/or a more superficial tubular uptake site. To test that hypothesis and to quantify the outward transfer of the filtered polypeptides, we estimated the tubular uptake pattern of the tracers in perfusion fixed rat kidneys after intravenous injection of *Cy and *Ap. METHODS: Autoradiographs were made from 10 mum thick slices of Microfil nephron casts from outer (OC), middle (MC) and inner (IC) cortical zones to quantify cortical border-crossing *Ap transfer. Single nephron glomerular filtration rate (snGFR) was estimated as the zonal uptake of *Ap corrected for *Ap transfer, divided by its time-integrated plasma concentration and the zonal number of glomeruli. RESULTS: *Ap and *Cy uptake fell exponentially along the proximal convoluted tubule (PCT), indicating an uptake proportional to luminal concentration. Uptake in IC exceeded that in MC and OC nephrons. The per cent PCT length with *Cy uptake (67.2 +/- 1.6) exceeded that of *Ap (54.6 +/- 1.8). The zonal border-crossing PCT length (29-34% of total PCT) from deep to more superficial cortical zones transferred 4-6% more *Cy than *Ap. CONCLUSION: Greater tubular uptake length of *Cy than of *Ap causes more cortical border-crossing of *Cy. The zonal snGFR estimated from Aprotinin uptake corrected for border-crossing agreed well with that obtained with the Hanssen ferrocyanide technique.

Pharmacokinetics and normal scintigraphic appearance of 99mTc aprotinin.

OBJECTIVE: To confirm the pharmacokinetics and biodistribution of 99mTc aprotinin in normal volunteers and to determine the optimum time for scanning post-injection, prior to further investigations of 99mTc aprotinin as an imaging agent for amyloidosis. METHODS: Five patients (three men and two women, average age 49 years, age range 38-66 years) without a history of amyloidosis or any of the associated diseases, were included in this prospective study. Blood and urine were collected and images were performed of the whole body and wrists. CONCLUSIONS: Normal biodistribution of 99mTc aprotinin includes early cardiac and lung activity in the blood pool phase with subsequent hepatic activity and renal excretion with variable splenic activity. There is variable bowel uptake on later images. The best quality images were obtained 90 min post-intravenous administration, and this is likely to be the optimum time for clinical imaging.

Role of the mucous/glycocalyx layers in insulin permeation across the rat ileal membrane.

The contribution of mucous/glycocalyx layers, as a diffusional or enzymatic barrier, to the absorption of insulin was investigated in situ and in vitro studies using rats. To remove the mucous/glycocalyx layers, ileal segments were exposed to a hyaluronidase solution in situ. The removal of the layers was confirmed by transmission electron microscopy, and the safety of the hyaluronidase pretreatment was established based on light microscopy, a constant mucosal membrane electrical resistance and the absence of lactate dehydrogenase leakage. In the in situ loop absorption experiment, hyaluronidase pretreatment significantly increased the plasma insulin level accompanied by an obvious hypoglycemic response. In the in vitro transport experiment, the apparent permeability coefficient of insulin was significantly increased by the hyaluronidase pretreatment, whereas that of 4.4 kDa fluorescein isothiocyanate-labeled dextran and of antipyrine, respective markers for passive para- and transcellular permeation, was unaffected. In the insulin degradation experiment in vitro, a significant amount of insulin was degraded in the compartment removed by hyaluronidase pretreatment. Thus, the mucous/glycocalyx layers functioned in insulin absorption as an enzymatic barrier and insignificantly affected diffusive absorption. In addition, co-administration of aprotinin, a protease inhibitor, further increased insulin absorption from ileum pretreated with hyaluronidase, implying the existence of another enzymatic barrier that influences insulin mucosal absorption.

Tubular absorption of filtered cystatin-C in the rat kidney.

As shown in previous studies, the two basic proteins aprotinin (Ap, 6.5 kDa) and cystatin (Cy, 13.3 kDa) can be used to estimate whole kidney glomerular filtration rate by measuring the renal cortical uptake relative to plasma concentration after i.v. injection. Local uptake of Ap can also be used to estimate local filtration rate, and the present experiments were undertaken to examine whether Cy would give a similar uptake pattern. Ap and Cy were labelled with 131I and 125I, respectively, and injected as an i.v. bolus. Frequent blood samples provided information on the filtered load. Five to 20 min after injection the kidney was clamped, frozen, and five tissue samples of 5-10 mg each were cut out from outer (OC), middle (MC) and inner cortex (IC) to be weighed and assessed for radioactivity. Five minute clearance ratios, Cy:Ap, were 1.36 +/- 0.04, 1.27 +/- 0.03 and 1.19 +/- 0.04 in OC, MC and IC, respectively. The higher Cy clearance was expected from a higher glomerular filtrate:plasma ratio of the less basic Cy (Donnan distribution). However, this does not explain the increase of Cy:Ap clearances going from IC to the OC. A surplus of extracellular uptake of Cy in superficial layers was excluded, leading to the following interpretation. In all cortical layers the proximal convoluted tubule, i.e. the protein uptake segment, is located more superficially than its parent glomerulus. A longer uptake segment for Cy than for Ap will therefore lead to a relative greater transfer of filtered Cy from IC to MC, and from MC to OC. Anatomical studies on single nephrons presented in another article lend strong support to this interpretation.

Variability of plasma aprotinin concentrations in pediatric patients undergoing cardiac surgery.

OBJECTIVES: Infants and children undergoing cardiopulmonary bypass for repair of congenital heart defects are at substantial risk for excessive bleeding, contributing greatly to morbidity and mortality. Aprotinin significantly reduces bleeding and transfusion requirements in adults but is of indeterminate value for pediatric patients. The aim of this study was to determine plasma aprotinin concentrations in these patients with a functional aprotinin assay. METHODS: Thirty patients less than 16 years of age scheduled for cardiac surgery with aprotinin were enrolled. Aprotinin was administered as a 25,000 KIU/kg bolus, 35,000 KIU/kg cardiopulmonary bypass prime, and 12,500 KIU.kg(-1).h(-1) continuous infusion. Blood samples for aprotinin concentrations (kallikrein-inhibiting units/milliliter) were obtained before aprotinin; 5 minutes post-bolus; 5 minutes after cardiopulmonary bypass initiation; 30 and 60 minutes on cardiopulmonary bypass; on discontinuation of aprotinin; 1 hour after aprotinin discontinuation; and 4 hours after permanent separation from cardiopulmonary bypass. For analysis, patients were grouped according to weight (<10 kg, 10-20 kg, >20 kg). Differences between weight groups were assessed using an exact test for categoric variables and 1-way analysis of variance for continuous variables. RESULTS: Aprotinin concentrations differed significantly across weight groups. Five minutes after aprotinin bolus and initiation of cardiopulmonary bypass, there was significant correlation between weight and aprotinin concentration (r =.57, P =.003; r =.69, P =.001, respectively). CONCLUSION: A functional assay reveals significant variability in aprotinin concentration for pediatric patients using current weight-based aprotinin dosing. Additional investigation is necessary to determine target aprotinin concentration dosing regimens to provide better efficacy.

Double liposomes: hypoglycemic effects of liposomal insulin on normal rats.

The biopharmaceutical characteristics of double liposomes (DLs) containing insulin were examined, and the usefulness of DLs in combination with aprotinin is discussed. Encapsulation of insulin was influenced by lipid composition, and the highest efficiency was observed with positively charged liposomes. Insulin encapsulated in liposomes, especially in DLs, was protected from enzymatic proteolysis. A portion of insulin molecules was adsorbed on the surface of the membrane when liposomes were prepared using a lipid with a positive charge and was degraded by enzymes. Remarkable hypoglycemic effects were observed after intragastric administration of DLs containing insulin at a dose of 20 IU/kg to normal male Wistar rats. The highest mean relative efficacy to administration was obtained with insulin-loading DLs containing aprotinin as a protease inhibitor. These results suggest that DLs are applicable as an oral dosage form for peptide drugs such as insulin etc., especially in combination with protease inhibitors.

Aprotinin uptake in the proximal tubules in the rat kidney I. Length of proximal tubular uptake segment.

Aprotinin (Ap), a basic polypeptide with a molecular weight of 6500, is filtered at the glomerular membrane without steric restriction and is completely absorbed by the proximal tubule cells. Here Ap is broken down to amino acids, but no breakdown products enter the peritubular circulation during the first 20 min following an intravenous injection. These properties have recently been exploited for measurement of local glomerular filtration rate, based on the assumption that the proximal tubular uptake site is located at the level of the filtering glomerulus. To evaluate that assumption we have now made serial autoradiographs of the rat kidney 20 min after intravenous injection of 2-750 microg of 125I-Aprotinin. With all doses the percent 125I-containing proximal tubular transections were about 50 in the outer and middle cortex and 35 in the inner third. We interpret these numbers to mean that all filtered Ap is taken up in the first two thirds of the proximal convoluted tubular length and does not reach the pars recta. Since the proximal tubule on average is located more superficial than its glomerulus, measurement of local Ap uptake will tend to overestimate glomerular filtration rate in outer layers of the cortex. Quantitative estimate of this "displacement" will be presented in a companion article.

Aprotinin uptake in the proximal tubules in the rat kidney. II. Uptake site relative to glomerulus.

Glomerular filtration rates in whole kidney and in outer, middle and inner cortical zones have previously been estimated by measuring the amount of iodinated Aprotinin, filtered and taken up in the first two thirds of the proximal convoluted tubules, in part positioned more superficial than the parent glomerulus. Thus, an appreciable amount of the absorbed Aprotinin may be located superficial to its filtration site and lead to an underestimate of glomerular filtration in deep cortical layers. Therefore, in this study we have measured the distance from the glomerulus to the center of proximal convoluted tubular ball and the site of Aprotinin uptake. Measurements were made on photos of Microfil-injected tubules and on camera lucida drawings of tubular transections from autoradiographs of nephrons containing both Microfil and iodinated Aprotinin. Both techniques showed that the center of the tubular ball was localized more superficial in all cortical layers. The average distance, in percent of cortical thickness, from all proximal convoluted tubular transections to the parent glomerulus was 9% in deep and 13% in middle and superficial cortex. Corresponding distances for tubular transections containing Aprotinin were 7 and 12%. Grain density in five reconstructed proximal convoluted tubules showed a continuous and exponential fall of Aprotinin along the uptake segment. The results may be used to estimate single nephron filtration rate from Aprotinin uptake and glomerular density in outer, middle, and inner cortex.