Auger Radiopharmaceutical Therapy Targeting Prostate-Specific Membrane Antigen.

UNLABELLED: Auger electron emitters such as (125)I have a high linear energy transfer and short range of emission (<10 µm), making them suitable for treating micrometastases while sparing normal tissues. We used a highly specific small molecule targeting the prostate-specific membrane antigen (PSMA) to deliver (125)I to prostate cancer cells. METHODS: The PSMA-targeting Auger emitter 2-[3-[1-carboxy-5-(4-(125)I-iodo-benzoylamino)-pentyl]-ureido]-pentanedioic acid ((125)I-DCIBzL) was synthesized. DNA damage (via phosphorylated H2A histone family member X staining) and clonogenic survival were tested in PSMA-positive (PSMA+) PC3 PIP and PSMA-negative (PSMA-) PC3 flu human prostate cancer cells after treatment with (125)I-DCIBzL. Subcellular drug distribution was assessed with confocal microscopy using a related fluorescent PSMA-targeting compound YC-36. In vivo antitumor efficacy was tested in nude mice bearing PSMA+ PC3 PIP or PSMA- PC3 flu flank xenografts. Animals were administered (intravenously) 111 MBq (3 mCi) of (125)I-DCIBzL, 111 MBq (3 mCi) of (125)I-NaI, an equivalent amount of nonradiolabeled DCIBzL, or saline. RESULTS: After treatment with (125)I-DCIBzL, PSMA+ PC3 PIP cells exhibited increased DNA damage and decreased clonogenic survival when compared with PSMA- PC3 flu cells. Confocal microscopy of YC-36 showed drug distribution in the perinuclear area and plasma membrane. Animals bearing PSMA+ PC3 PIP tumors had significant tumor growth delay after treatment with (125)I-DCIBzL, with only 1 mouse reaching 5 times the initial tumor volume by 60 d after treatment, compared with a median time to 5 times volume of less than 15 d for PSMA- PC3 flu tumors and all other treatment groups (P = 0.002 by log-rank test). CONCLUSION: PSMA-targeted radiopharmaceutical therapy with the Auger emitter (125)I-DCIBzL yielded highly specific antitumor efficacy in vivo, suggesting promise for treatment of prostate cancer micrometastases.

Diagnostic ultrasound activates pure prekallikrein.

Diagnostic ultrasound activates the contact phase of human coagulation. This has been seen in human blood or plasma or with purified factor 12. The present work aimed to quantify a possibly triggering action of ultrasound on purified prekallikrein, the second of the two main triggers of the intrinsic hemostasis cascade. Either 2.7 µg/ml human prekallikrein or for control 1 µg/ml kallikrein in 26% glycerol - 0.54% NaCl-10.6 mmol/l Na3 citrate pH 7.4, in emptied polypropylene coagulation monovettes (Sarstedt) were exposed to diagnostic ultrasound (Siemens Acouson Antares, 5 MHz, 0.6 TIB, 0.6 TIS) for 0-5 min at room temperature (RT). Fifty microliter samples were withdrawn in duplicate and placed into an U-wells high quality microtiter plate (Brand 781600). Then 10 µl 2 mmol/l chromogenic substrate HD-CHG-Ala-Arg-pNA in 0.45% NaCl were added, and the increase in absorbance with time (ΔA405 nm /t at 37°C) was determined by a microtiterplate photometer with a 1 mA resolution (PHOmo; anthos). Exposure to diagnostic ultrasound biphasically increased the chromogenic activity of a prekallikrein solution in 26% glycerol. About 3-4 min ultrasound at 23 °C generated about 0.02 µg/ml kallikrein, that means that about 1% of pure prekallikrein in glycerol was converted into kallikrein. Thus, diagnostic ultrasound activates purified human prekallikrein to kallikrein. The ultrasound energy seems to fold the latent proenzyme prekallikrein into the active enzyme kallikrein. This contributes to explain the triggering action of ultrasound on the contact system of plasmatic human coagulation. Conversion of only 1% of prekallikrein into kallikrein is absolutely sufficient to start the intrinsic coagulation cascade. The clinical consequence of this action of ultrasound on intrinsic coagulation is that patients at risk for thrombosis, for example, patients with insufficiencies of hepatocytes, AT-3, C1-ina, or fibrinolysis should be protected by low-molecular-weight-heparin prior to the exposure of ultrasound, especially upon its prolonged exposure.

Effects of ionizing irradiation and beta-adrenergic stimulation on gene expression pattern in rat submandibular glands.

Radiotherapy administrated to patients with head and neck malignancies and prior to bone marrow transplantation often results in severe xerostomia. We evaluated the expression of early response proto-oncogenes (c-fos and jun B), tissue specific genes (proline rich protein [PRP] and kallikrein), and proteolysis linked utiquitin gene following exposure to 15 Gy irradiation alone or in combination with beta-adrenergic stimulation of the rat submandibular glands. Head and neck irradiation resulted not only in dysfunction and tissue loss of the salivary glands but also in a systemic effect expressed as profound body weight loss. Irradiation alone was found to induce expression of the jun B but not the c-fos proto-oncogenes. The combination of irradiation and beta-adrenergic stimulation by isoproterenol induced earlier expression of jun B and profound expression of the c-fos proto-oncogene in comparison to irradiation alone. In contrast, the kallikrein and ubiquitin genes were expressed constitutively and were not affected by irradiation alone or in combination with beta-adrenergic stimulation. In addition, irradiation had no effect on submandibular gland mRNA translation. We observed that the expression of the genes whose regulation is associated with DNA damage (i.e. jun B and c-fos) was enhanced by irradiation alone or in combination with isoproterenol administration. In contrast, the expression of genes associated with the routine functional integrity of the cell (i.e. kallikrein, ubiquitin, and PRP) was unaffected. These findings, in addition to delayed gland dysfunction, leads us to believe that the irradiation induced injury to the submandibular glands is to be attributed to reproductive stem cell death which may be partly obliterated in the clinical setting by better understanding.

The effect of local radiotherapy on kallikrein activity in saliva secreted by parotid gland in patients with head and neck cancers.

The samples of parotid gland saliva, collected from control human subjects and those taken from patients with head and neck cancers were submitted to the assay of protein concentration and kininogenase and amidolytic kallikrein activities. No patients with parotid gland tumours were included. The effect of pilocarpine stimulation on these parameters was studied. It was found that the saliva secreted by parotid gland of the investigated patients contains less protein and lower kininogenase activity in comparison to control subjects. Pilocarpine administration resulted in an increase of protein concentration and a decrease of kallikrein activity both in control and investigated subjects. Radiotherapy did not evoke any significant changes in spontaneously secreted saliva. The radiotherapy resulted in a progressive decrease of protein concentration and kallikrein activity in saliva secreted by pilocarpine stimulated glands. The kallikrein activity per mg of protein contained in spontaneously secreted saliva increased significantly during radiotherapy but it distinctly decreased in the saliva of pilocarpine-treated patients. It allows to conclude that the parotid glands do not lose their ability to synthesize and secrete kallikrein during radiotherapy of head and neck cancers.

Inactivation of viruses during ultraviolet light treatment of human intravenous immunoglobulin and albumin.

A comparison of ultraviolet (UV) irradiation of two wavelength ranges UVB (280-320 nm) and UVC (lower than 280 nm) showed that UVC in particular could very effectively inactivate, in intravenous immunoglobulin (IVIG) and albumin preparations, non-enveloped and non-acid labile model viruses (i.e., Polio 2 and T4 phage) and dry heat-resistant viruses (vaccinia and T4 phage). This effective virucidal treatment (5 min, 5,000 J/m2 dose) was achieved before an unacceptable level of IVIG aggregates occurred. The use of UV irradiation to inactivate infectious agents could add safety and supplement current methods, e.g. solvent/detergent, low pH, which do not inactivate non-enveloped, non-acid labile or dry-heat-resistant viruses at present.

Inactivation of kallikrein and kininases and stabilization of whole rat brain kinin levels following focused microwave irradiation.

Focused microwave irradiation was employed to stabilize endogenous whole rat brain bradykinin levels prior to a simple extraction procedure. Skull microwave exposure (2450 MHz, 3.8 kW., 2.45 sec) resulted in inactivation to less than 5% of control of whole brain kallikrein and kininase activity. Using this adequate exposure duration whole rat brain kinin levels as measured by a sensitive radioimmunoassay were approximately 0.6 pmol/g (wet weight). Further purification of irradiated brain extracts using HPLC revealed that immunoreactive kinin eluted as a single peak that co-chromatographed with authentic bradykinin. Microwave fixation duration of 1.25 sec yielded greatly increased levels of immunoreactive kinin which following HPLC purification eluted in two peaks, corresponding to authentic bradykinin and T-kinin, respectively. The tissue injury resulting from incomplete microwave fixation resulted in the release of kinins. This excess immunoreactive kinin may be derived from cerebral blood, since the predominant form of kinin-generating protein in plasma is T-kininogen.

The level of blood kinins, their creation and inactivation after one exposure to intense ultraviolet radiation in rabbits.

Experimental rabbits were exposed to ultraviolet radiation once for 45 minutes, and blood samples were obtained from the carotid artery in these animals 45 min and 3, 6 and 24 hours after the end of this exposure. In the group of control rabbits blood samples were obtained in the same way without previous exposure to radiation. The hairs on the back were cut closely at the skin and this skin area was exposed to ultraviolet rays from a Hanau Q 400 burner at 405--289 nm wavelengths and at an intensity of 134 000 erg/sec/cm2, using an UG 2 T Schott filtre and an absorber of long-wave radiation. Blood samples were taken under thiopental anaesthesia. In the samples the level of free kinins was determined in the blood, and the level of kininogen and the activity of kallikreins and kininases were determined in the plasma. In the irradiated animals a rise of the kinin level was observed, with a fall in the kininogen level most pronounced after 3 hours, while the activity of kallikreins was raised and that of kininases was decreased particularly after 6 hours.

Effect of long-term exposure of rabbits to ultraviolet radiation on the level, creation and inactivation of kinins in blood.

Experimental rabbits were exposed to ultraviolet radiation during 6 weeks once daily for 10 minutes (from a high-pressure. Hanau Q 400 mercury lamp with a Schott UG 2 T filtre and absorber of long-wave radiation using ultraviolet rays of 405--289 nm wavelength and 134 000 erg/sec/cm2 power, directed onto skin with cropped hair of the back). Under general anaesthesia with pentothal blood samples were obtained from the carotid artery in 4 groups of 8 rabbits in each group. The blood samples were taken from non-exposed control rabbits and from the experimental groups after 2, 4 and 6 weeks of exposure. In the samples the levels of free kinins in the blood, and kininogen, and the activity of kallikreins and kininases in the plasma were determined. In the irradiated animals a progressive rise of free kinins most pronounced after 6 weeks was observed, and other findings included: a fall of kininogen level particularly steep after 2 weeks, very small rise in the activity of kallikreins, and progressing reduction of the activity of kininase, particularly steep after 2 weeks.