Design, Synthesis, Computational, and Preclinical Evaluation of natTi/45Ti-Labeled Urea-Based Glutamate PSMA Ligand.

Despite promising anti-cancer properties in vitro, all titanium-based pharmaceuticals have failed in vivo. Likewise, no target-specific positron emission tomography (PET) tracer based on the radionuclide 45Ti has been developed, notwithstanding its excellent PET imaging properties. In this contribution, we present liquid-liquid extraction (LLE) in flow-based recovery and the purification of 45Ti, computer-aided design, and the synthesis of a salan-natTi/45Ti-chelidamic acid (CA)-prostate-specific membrane antigen (PSMA) ligand containing the Glu-urea-Lys pharmacophore. The compound showed compromised serum stability, however, no visible PET signal from the PC3+ tumor was seen, while the ex vivo biodistribution measured the tumor accumulation at 1.1% ID/g. The in vivo instability was rationalized in terms of competitive citrate binding followed by Fe(III) transchelation. The strategy to improve the in vivo stability by implementing a unimolecular ligand design is presented.

Turnover of 125I-labelled tissue kallikrein following intraduodenal or intravenous administration.

UNLABELLED: Tissue kallikrein is released in the body both physiologically and in many inflammatory disorders. Little is, however, known about the turnover of released tissue kallikrein in humans. Approximately 1 mg of tissue kallikrein (mol wt 43,000 Da) was purified from 85 L human urine by: (1) ultracentrifugation, (2) filtration through an aprotinin-coupled Sepharose 4B column, followed by (3) gel filtration over a Sephadex G-75 column. The elimination, after intraduodenal or intravenous administration of purified tissue kallikrein radiolabelled with 125I, was followed by collecting serial samples of plasma, urine and faeces from three volunteers. Within 72 h, about 96% of the intraduodenally administered radioactivity had been excreted in urine, and approximately 5.4% in faeces, mainly as 125I. No intact 125I-tissue kallikrein was found in plasma, urine or faeces after the intraduodenal instillation of the protein. The plasma half-life of 125I-tissue kallikrein up to 3 h after intravenous injection was 9 min and, thereafter, 20 h. The 125I-tissue kallikrein was quickly bound to a plasma protein with a mol wt of about 67 kDa, but some of the radioiodinated tissue kallikrein was still unbound 15 min after injection, judged by gel filtration on Sephadex G-200 columns. Most of the radioactivity was excreted in the urine as 125I, but about 4-6% was recovered as free 125I-tissue kallikrein. CONCLUSION: The use of tissue kallikrein as an oral drug appears, therefore, to be useless. Tissue kallikrein released into plasma seems to be quickly bound to a protein with a mol wt of 67 kDa, probably kallistatin or Protein C inhibitor, but some tissue kallikrein seems to be unbound and may have some physiological or pathophysiological action. The unbound tissue kallikrein is, at least partly, cleared from the circulation by the kidneys, and tissue kallikrein in the urine may partly be derived from plasma.

Plasma-kallikrein clearance during liver regeneration after partial hepatectomy in the rat.

AIMS/BACKGROUND: The liver clears circulating plasma-kallikrein through a receptor-mediated endocytosis process: an initial fast phase is followed by a slow exponential phase. METHODS: To determine whether the clearance rate of plasma-kallikrein is affected during liver regeneration, we perfused isolated rat livers with rat plasma-kallikrein (rPK) at 0, 1, 2, 3 and 7 days after partial hepatectomy or sham operation. RESULTS: Liver regeneration was followed by the expression of the proliferating-cell nuclear antigen (PCNA) labeling index. The serum concentration of alpha2-macroglobulin, an acute phase protein in rats, was measured. At day 1, the fast phase of rPK clearance rate increased in hepatectomized rats when compared with day 0 (4.9+/-0.4 and 3.7+/-0.4 mU/g liver min, p<0.05). However, at day 2, the rPK fast phase clearance rate dropped significantly (2.6+/-0.2, p<0.05), when compared with day 1. No difference was found among the sham groups at different days of hepatectomy. These changes seem to be independent of the acute phase reaction. The regenerative liver weight increased continuously during the observation period. PCNA expression increased significantly after hepatectomy, with maximal PCNA-labeling indices at days 1 and 2, declining thereafter. CONCLUSION: The rPK fast phase clearance rate changes during liver regeneration, with a zenith occurring when PCNA labeling index is maximal (day 1) and a nadir occurring at the mitotic phase (day 2).

The fate of plasma kallikrein in normal and kininogen-deficient rats.

The clearance of exogenous plasma kallikrein, its uptake by liver, spleen, kidneys, lungs and its extravasation in the paws were determined in normal Wistar rats, normal and kininogen-deficient Brown Norway rats. Kallikrein was purified from rat plasma and labelled with 125I. After intravenous injection of 125I-kallikrein, the disappearance of acid-precipitable kallikrein from the blood fits a biexponential curve similar in the three groups of rats: a rapid initial clearance (T1/2 around 3 min) followed by a phase of slower elimination (T1/2 around 50 min). Removal of kallikrein from the blood was associated with a large uptake of radioactivity by the liver: 67% of the 125I-kallikrein cleared from the blood at 10 min. The kidneys and the spleen accumulated small amounts of the radioactivity. The uptake of kallikrein by the spleen was slightly reduced in kininogen-deficient rats. The kininogen deficiency in Brown Norway rats from the strain BN/May Pfd was confirmed by the low levels of kinins released by tissue kallikrein and by a prolongation of activated thromboplastin times in the plasma of these animals. We concluded that plasma kallikrein is rapidly cleared from the circulation of the rat. The liver is the main clearing organ of plasma kallikrein. The disappearance of kallikrein from the circulation is not affected by the lack of high molecular weight kininogen, except in the case of the uptake of the enzyme performed by the cells of the spleen, which is reduced.

Tópicos farmacológicos atuais sobre as cininas na clínica / Actual pharmacologic topics about kinins in clinic

Os autores destacam os conhecimentos básicos sobre o sistema calicreínacinina, abordando seus aspectos bioquímicos fundamentais e evidenciando sua relevância farmacológica na prática médica atual

High level of circulating human tissue kallikrein induces hypotension in a transgenic mouse model.

We established a unique transgenic mouse model in liver-targeted expression of human tissue kallikrein using a mouse albumin enhancer and promoter. Northern blot analysis and ELISA showed that human tissue kallikrein was predominantly expressed in the liver of transgenic mice and secreted into the circulation at a high level. The transcript was also detected in the kidney, pancreas, salivary gland and heart at a low level by reverse transcription-polymerase chain reaction followed by Southern blot analysis. Systolic blood pressures were measured by the tail-cuff method, all three independent transgenic mouse lines are hypotensive (84.6 +/- 1.0 mmHg, n = 17; 84.5 +/- 1.5 mmHg, n = 9; 83.1 +/- 0.8 mmHg, n = 13, P < 0.01) compared with the control mice (100.9 +/- 0.9 mmHg, n = 17). Administration of aprotinin, a potent tissue kallikrein inhibitor or Hoe 140, a bradykinin receptor antagonist, restored the blood pressure of transgenic mice but had no significant effect on control littermates. These studies show that over-production of tissue kallikrein in the circulation plays a role in blood pressure regulation.

Pharmacokinetic studies of human urinary kininogenase in healthy volunteers and animals.

Plasma concentrations of human urinary kininogenase (HUK) were determined in healthy volunteers during and after intravenous (iv) infusion by enzyme immunoassay (EIA). Plasma kinin concentrations were also determined by radioimmunoassay (RIA), and related to HUK concentrations. When HUK was infused [at 0.04, 0.075, 0.15, and 0.3 p-nitroaniline unit (PNAU)/body] over 30 min, plasma HUK concentration rapidly increased and reached a maximum at the end of dosing. Then, the concentration of HUK in plasma decreased biexponentially, and the elimination half-life of the terminal phase was found to be approximately 170 min. The area under the curve of concentration versus time from 0 to 180 min (AUC0-180min) and the maximum concentration (Cmax) increased in proportion to the dose, whereas the pharmacokinetic parameters [mean residense time (MRTinf) = 200-270 min, plasma clearance (CLp) = 2.5-3.3 mL/min/kg, volume of distribution at steady state (Vdss) = 470-730 mL/kg] did not very significantly within the dose range of the present study. On the other hand, when HUK was infused (at 0.15 PNAU/body), plasma kinin concentrations reached approximately 2 ng of bradykinin eq/mL 15 min after the onset of administration. This concentration was maintained during the dosing period, after which kinin was rapidly eliminated, and its concentration returned to baseline at 10 min after dose withdrawal. Plasma kinin concentrations at 15 to 30 min after the onset of dosing (at 0.075, 0.15, and 0.3 PNAU/body) increased in proportion to the dose. The pharmacokinetic parameters of HUK (MRTinf, CLp, Vdss) were compared with those of rats, rabbits, and dogs (log-log plots of body weight versus MRTinf, CLp, and Vdss). The Vdss value showed a good correlation (r = 0.996 for n = 4) with the body weight of respective animal species, the correlation with CLp was weak (r = 0.911), and MRTinf did not exhibit any correlation.

The catabolism of intact, reactive centre-cleaved and proteinase-complexed C1 inhibitor in the guinea pig.

Clearance rates in the guinea pig were determined for intact guinea pig and human C1 inhibitor, the complexes of both inhibitors with human Cls, beta factor XIIa and kallikrein, and for each inhibitor cleaved at its reactive centre with trypsin. Intact human and guinea pig C1 inhibitor were cleared from the circulation more slowly (t1/2s of 9-7 h and 12.1 h and fractional catabolic rates (FCRs) of 0.09 and 0.117) than any of their cleaved or complexed forms. The reactive centre-cleaved inhibitors were cleared with half-lives of 6.75 h for humans and 10.1 h for the guinea pig. The complexes with target proteases were catabolized much more rapidly, with half-lives ranging from 3-08 h to 4.3 h. The complexes with kallikrein were cleared more slowly than those with Cls and beta factor XIIa. Complexes prepared with the guinea pig and human inhibitors were cleared at equivalent rates. The free inactivated proteases were cleared at rates similar to the equivalent complexes, except for kallikrein, which was cleared more rapidly than its complex. The fact that the complexes with different target proteases differed in their catabolism and that protease and complex catabolism were similar suggests that protease may play a direct role in clearance.

Kinetic characterization of rat tissue kallikrein using N alpha-substituted arginine 4-nitroanilides and N alpha-benzoyl-L-arginine ethyl ester as substrates.

Hydrolysis of seven N alpha-substituted L-arginine 4-nitroanilides: benzoyl-arginine p-nitroanilide (Bz-Arg-Nan), tosyl-arginine p-nitroanilide (Tos-Arg-Nan), acetyl-leucyl-arginine p-nitroanilide (Ac-Leu-Arg-Nan), acetyl-phenylalanyl-arginine p-nitroanilide (Ac-Phe-Arg-Nan), benzoyl-phenylalanyl-arginine p-nitroanilide (Bz-Phe-Arg-Nan), tosyl-phenylalanyl-arginine p-nitroanilide (Tos-Phe-Arg-Nan), and D-valyl-leucyl-arginine p-nitroanilide (D-Val-Leu-Arg-Nan), and the N alpha-substituted L-arginine ester: benzoyl-arginine ethyl ester (Bz-Arg-OEt), by rat tissue kallikrein was studied throughout a wide range of substrate concentrations. The enzyme showed a bimodal behavior with all the substrates tested except Tos-Arg-Nan. At low substrate concentrations (10 to 170 microM for p-nitroanilides and 50 to 190 microM for Bz-Arg-OEt) the hydrolysis followed Michaelis-Menten kinetics, but at higher substrate concentrations (150 to 700 microM for p-nitroanilides and 200 to 1800 microM for Bz-Arg-OEt) a deviation from Michaelis-Menten kinetics was observed with a significant decrease in hydrolysis rates. At high concentrations of the p-nitroanilide substrates, partial enzyme inhibition was observed, whereas complete enzyme inhibition was observed with Bz-Arg-OEt at high concentration. The kinetic parameters reported here were calculated from data for substrate concentrations range where the enzyme followed Michaelis-Menten behavior. D-Val-Leu-Arg-Nan (Km = 24 +/- 2 microM; Vmax = 10.42 +/- 0.28 microM/min) was the best substrate tested, followed by Ac-Phe-Arg-Nan (Km = 13 +/- 2 microM; Vmax = 3.21 +/- 0.11 microM/min), while Tos-Arg-Nan (Km = 29 +/- 2 microM; Vmax = 0.10 +/- 0.002 microM/min) was the worst of the tested substrates for rat tissue kallikrein. For the hydrolysis of Bz-Arg-OEt (Km = 125 +/- 15 microM; Vmax = 121.3 +/- 7.6 microM/min), the kinetic parameters using a substrate inhibition model can reasonably account for the observed enzyme behavior, with a Ksi value about 13.6 times larger than the estimated Km value.

Kinetic characterization of rat tissue kallikrein using NÓ-substituted arginine 4-nitroanilides and NÓ-benzoyl-L-arginine ethyl ester as substrates

Hydrolysis of seven N(alpha-substituted L-arginine 4-nitroanilides: henzoyl-arginine p-nitroanilide (Bz-Arg-Nan), tosyl-arginine p-nitroanilide (Tos-Arg-Nan), acetyl-leucyl-arginine p-nitroanilide (Ac-Leu-Arg-Nan), acetyl-phenylalanyl-arginine p-nitroanilide (Ac-Phe-Arg-Nan), benzoyl-phenylalanyl-arginine p-nitroanilide (Bz-Phe-Arg-Nan), tosyl-phenylalanyl-arginine p-nitroanilide (Tos-Phe-Arg-Nan), and D-valyl-leucyl-arginine p-nitroanilide (D-Val-Leu-Arg-Nan), and the N(alpha-substituted L-arginine ester: benzoyl-arginine ethyl ester (Bz-Arg-OEt), by rat tissue kallikrein was studied throughout a wide range of substrate concentrations. The enzyme showed a bimodal behavior with all the substrates tested except Tos-Arg-Nan. At low substrate concentrations (10 to 170 muM for p-nitroanilides and 50 to 190 muM for Bz-Arg-OEt) the hydrolysis followed Michaelis-Menten kinetics, but at higher substrate concentrations (150 to 700 muM for p-nitroanilides and 200 to 1800 muM for Bz-Arg-OEt) a deviation from Michaelis-Menten kinetics was observed with a significant decrease in hydrolysis rates. At high concentrations of the p-nitroanilide substrates, partial enzyme inhibition was observed, whereas complete enzyme inhibition was observed with Bz-Arg-OEt at high concentration. The kinetic parameters reported here were calculated from data for substrate concentrations range where the enzyme followed Michaelis-Menten behavior. D-Val-Leu-Arg-Nan (Km = 24 ñ 2 muM; Vmax 10.42 ñ 0.28 muM/min) was the best substrate tested, followed by Ac-Phe-Arg-Nan (Km = 13 ñ 2 muM; Vmax = 3.21 ñ 0.11 muM/min), while Tos-Arg-Nan (Km = 29 ñ 2 muM; Vmax, = 0. 10 ñ 0.002 muM/min) was the worst of the tested substrates for rat tissue kallikrein. For the hydrolysis of Bz-Arg-OEt (Km = 125 ñ 15 muM; Vmax = 121.3 ñ 7.6 muM/min), the kinetic parameters using a substrate inhibition model can reasonably account for the observed enzyme behavior, with a Ksi value about 13.6 times larger than the estimated Km value.

Involvement of renal kallikrein in the regulation of bicarbonate excretion in rats.

1. The experiments reported here were performed to test the hypothesis that renal kallikrein is involved in the regulation of acid-base balance. 2. The bicarbonate concentration and the kallikrein activity in the spontaneously voided urine of conscious rats (experiment 1) were inversely correlated (correlation coefficient (r) = -0.63, P < 0.0001). The correlation was even greater when the urinary bicarbonate concentration was expressed per milligram excreted creatinine (r = -0.74, P < 0.00002). 3. Intravenous injection of the kallikrein inhibitor aprotinin in barbiturate-anaesthetized rats (experiment 2) reduced urinary kallikrein activity (P < 0.05) and increased bicarbonate excretion rate (P < 0.012). 4. Renal arterial infusion of aprotinin in barbiturate-anaesthetized rats (experiment 3) reduced urinary kallikrein activity (120 min, P < 0.01), and increased bicarbonate excretion rate (120 min, P < 0.01). Animals infused with the inhibitor developed a moderate metabolic acidosis (base excess: control, 2.9 +/- 0.7 mM (mean +/- S.E.M.); experimental, -8.1 +/- 0.7 mM; P < 0.05). 5. The bicarbonate concentration of urine fractions obtained after retrograde injection of kallikrein through the ureter into the collecting duct system of barbiturate-anaesthetized rats was lower than that from kidneys administered the vehicle (experiment 4; P < 0.001). A retrograde injection of bradykinin was without effect (experiment 5). 6. We conclude that renal kallikrein is involved in the regulation of urinary bicarbonate excretion. Increased intraluminal activity of the enzyme reduces, and decreased kallikrein activity increases, bicarbonate excretion. The enzyme may be a component of a negative feedback loop controlling the hydrogen ion activity of the extracellular space.

In vivo catabolism of human kallikrein-binding protein and its complex with tissue kallikrein.

We recently identified and purified a novel human kallikrein-binding protein (HKBP) from human plasma. The HKBP forms a 92 kd sodium dodecyl sulfate-stable and heat-stable complex with tissue kallikrein. This study was undertaken to characterize the plasma clearance and tissue distribution of exogenously administered HKBP and its complex with tissue kallikrein. Human tissue kallikrein was first incubated with purified HKBP, and the high-molecular-weight complex was separated from unbound proteins on a high-pressure liquid chromatography gel filtration column. Tissue kallikrein, kallikrein-binding protein, and their complex were labeled with iodine-125 and then injected intravenously into Sprague-Dawley rats. The disappearance rates of trichloracetic acid-precipitable radioactivity from the circulation were determined. The clearance profile of HKBP shows a nonlinear pattern with an apparent half-life of 65 minutes (n = 4). The plasma clearance of HKBP complexed with kallikrein shows a similar profile but a shorter half-life of 33 minutes (n = 3). HKBP and its complex with kallikrein were mainly taken up by the liver but to a lesser degree by the kidney, lung, and other tissues. Labeled human kallikrein has an apparent half-life of 8 minutes (n = 4), and its clearance consists of a fast and a slow component. The data indicate that kallikrein-HKBP complex is cleared from the circulation two times faster than that of the binding protein alone and that it persists in the circulation four times longer than kallikrein alone. The results support the notion that more than one pathway exists for the metabolism of tissue kallikrein and that HKBP plays a role in modulating tissue kallikrein's bioavailability.

Absorption studies with porcine pancreatic kallikrein in man.

Porcine pancreatic kallikrein (PPK), the main component of Padutin (Bayropharm, FRG), is used since 1974 for the treatment of some forms of idiopathic infertility in man. During kallikrein therapy the number of spermatozoa increases, and qualitative and quantitative sperm motility is improved. In order to investigate the intestinal absorption of PPK in man a clinical study with 7 healthy volunteers was performed. 4500 KE (corresp. to 2.8 mg PPK) and 600 KE (corresp. to 0.38 mg PPK) of kallikrein respectively was orally administered in one dosis. Serum and urine samples were collected several times within 24 hours. Seminal plasma was collected 4-5 days before and 8 hours after treatment with kallikrein. Absorbed PPK was determined using a highly sensitive bioluminescence-enhanced enzyme immunoassay (1) and a newly developed light measuring equipment (MTP reader) (3). The limit of detection was 1 pg/ml in serum corresponding to 6 amol PPK per assay. For both groups (600 KE and 4500 KE) absorption of PPK in serum was found. Maximum absorption was observed between 4 and 12 h and between 2 and 6h, respectively, after oral application of kallikrein. Renal excretion plays no important role in the elimination of PPK from serum. In gel filtration experiments with blood samples of volunteers in which absorbed PPK was detected, one peak of immunochemically active material corresponding to a molecular mass of 82 kDa was found. According to our results, kallikrein is absorbed in unaltered form by the intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue kallikrein clearance by the liver--effect of plasma components on kallikrein uptake by cultured rat hepatocytes.

Rat urinary kallikrein (RUK) injected intravenously to the rat rapidly disappeared from the blood circulation and mainly distributed to the liver. RUK was also incorporated to the primary cultured rat hepatocytes. The amounts of RUK incorporated into the hepatocytes increased by preincubation of RUK with rat plasma. These results and SDS-PAGE analysis suggested some plasma components may be involved in tissue kallikrein clearance in the blood.

Lack of oral kallikrein in lowering systemic blood pressure in primary hypertension.

Primary hypertension is associated with a lack in renal kallikrein activity which might be one of the reasons for the blood pressure elevation. Some smaller and partially uncontrolled studies suggested that an oral substitution of glandular kallikrein lowers blood pressure by a kinin-mediated vasodilation and increased natriuresis. To test this hypothesis we treated in two studies over 100 patients with untreated mild to moderate primary hypertension (WHO I-II) for 5 resp. 12 weeks in a double blind randomized and placebo controlled manner with 1800 U glandular kallikrein orally. Blood pressure measurements were performed according to the two study designs after 3 and 5 resp. 8 and 12 weeks of treatment sphymomanometrically in the day time course. No significant changes in blood pressure by kallikrein treatment could be observed at any time. Neither renal kallikrein excretion, renin and ACE-activity nor blood glucose concentration in diabetics or non-diabetics was changed. Thus, we could undoubtedly demonstrate that oral applied glandular kallikrein has no effect on primary hypertension.

[Absorption of swine pancreatic kallikrein in humans]. / Resorptionsstudie mit Schweinepankreas-Kallikrein am Menschen.

Porcine pancreatic kallikrein (PPK), the main component of Padutin, has been used in andrology since the beginning of the seventies for the treatment of oligozoospermia and asthenozoospermia. During kallikrein therapy the number of spermatozoa increases, and qualitative and quantitative sperm motility is improved. In order to investigate the intestinal absorption of PPK in man, a clinical study in 26 healthy volunteers was performed. Four of them were given single oral doses of 4500 KU (kallikrein units), the remaining 22 subjects received 600 KU. Serum and urine samples were collected several times within 24 h. Seminal plasma was collected 4-5 days before and 8 h after kallikrein therapy. Absorbed PPK was determined using a highly sensitive bioluminescence-enhanced enzyme immunoassay and a newly developed light-measuring equipment (MTP-reader). In both groups (600 KU and 4500 KU) absorption of PPK in serum was found. The minimum absorption rates were 0.82% and 0.43%, respectively, of the administered PPK, corresponding to an amount of 5 KU per subject. Renal excretion was determined to be 0.27% of the absorbed kallikrein and thus plays only a secondary role in the elimination of PPK from the blood. Moreover, by gel filtration experiments it could be demonstrated that PPK is absorbed in unaltered form and is slowly inhibited in the serum, possibly by a1-proteinase inhibitor. According to our results, kallikrein is absorbed in unaltered form by the intestine. The absorbed amount of PPK is sufficient to exert a possible effect in enzymatically active form on the target cells in the male gonads for several hours.

Chronic administration of ethamol does not alter plasma kallikrein hepatic clearance

1. The clearance of plasma kallikrein bu the isolated and perfused liver of rats chronically intoxicated with ethanol was studied. Alcohol was added to the diet as 36% of total calories, and the animals were kept on this diet for 5-7 weeks. 2. The hepatic clearance of plasma kallikrein by these rats (uptake half-life, 15 ñ 2 min; N = 3) was similar to that observed in the control groups (normal diet, iptake half-life, 14 ñ 2 min; N = 5, or normal diet with sucrose added as 36% of total calories, uptake half-life, 16 ñ 3 min; N = 4). 3. These results provided indirect evidence that the endocystosis mechanism of plasma kallikrein by the liver differs from that descrived for glycoproteins which use the galactosyl receptor, since liver endocystosis via this latter system is reduced by chronic alcohol intoxication