A formulation of pancreatic pro-enzymes provides potent anti-tumour efficacy: a pilot study focused on pancreatic and ovarian cancer.

Proteolytic enzymes have shown efficacy in cancer therapy. We present a combination of the two pro-enzymes Trypsinogen and Chymotrypsinogen A with potent in vitro and in vivo anti-tumour efficacy. A synergetic anti-tumour effect for Trypsinogen and Chymotrypsinogen A was determined at a ratio 1:6 (named PRP) using 24 human cancer cell lines. The antiangiogenic effect of PRP was analysed by matrigel-based tube formation and by fibrous capsule formation assays. Furthermore, cell invasion and wound healing assays together with qRT-PCR determination of epithelial-to-mesenchymal transition (EMT) markers were performed on human cancer cells treated with PRP. Additionally, in vivo pharmacokinetic studies were implemented and the PRP's anti-tumour efficacy was explored against orthotopic pancreatic and ovarian cancer tumours. PRP formulation was proven to inhibit in vitro angiogenesis, tumour growth, cancer cell migration and invasiveness; and to be an effective and well tolerated in vivo anti-tumour treatment. Finally, the clinical efficacy of a suppository formulation containing both pancreatic pro-enzymes in the context of a UK Pharmaceuticals Special Scheme was evaluated in advanced cancer patients. Consequently, PRP could have relevant oncological clinical applications for the treatment of advanced or metastatic pancreatic adenocarcinoma and advanced epithelial ovarian cancer.

A Compelling Case for the Use of Perioperative Zymogen Protein C for Increased Patient Safety.

It is imperative to maintain normal blood flow to provide adequate oxygen supply to specific organs and cells, as well as for the removal of metabolic byproducts. Therefore, any situation that results in blood clotting can injure or kill living tissues. In this paper, we describe a case where a protein C deficient subject who would, by all medical indicators, be at 100 % risk of experiencing thrombophlebitis, deep vein thrombosis, and or lung emboli, is able to escape all pathologies by using perioperative zymogen protein C (ZPC). This protein C deficient patient has a long history of blood clotting, particularly from surgical procedures. The patient is 81 years old and first experienced clotting due to hernia surgery in 1964, when he was hospitalized for 16 days post-surgery with life threatening complications. It was later determined in 1980, after many episodes, that the patient had hereditary protein C deficiency at the 38 % level. In his hernia surgery, perioperative ZPC was used along with accepted anticoagulation procedures with no blood clots or other related side effects occurring. This procedure can greatly benefit protein C deficient patients, and could potentially find use for non-PC deficient patients in surgeries and a variety of other medical treatments. This particular case helps to validate the importance of ZPC in effecting safer surgery in high-risk patients. It also supports the mechanism of ZPC acting as an anticoagulant without causing bleeding. Most importantly, each clinical case study represents a unique combination of surgeon, hematologist, medical staff, and patient functioning as a coordinated team. In this case, smaller amounts of very expensive ZPC achieved safe and efficacious results, which is hugely important for future clinical applications when considering the production cost of ZPC. More studies must be done to establish minimum dosing while achieving safe and efficacious outcomes.

Pancreaticoduodenectomy using perioperative zymogen protein C to help prevent blood clotting: a trilogy on increased patient safety.

The blood clotting mechanism is a very important and complex physiologic process. Blood flow must be continuous through the blood vessels to provide essential oxygen and nutrients to the cells of the body. Dr. Melvin H. Knisely (Honorary First President of ISOTT, 1973) named and pioneered research in blood sludging and clotting which led to his nomination for the Nobel Prize by Dr. August Krogh in 1948. Abnormal clotting is a pathological state that can inhibit and prevent normal blood flow, leading to reduced oxygen transport to tissue from the microcirculation. It can result in the death of cells and tissues, including entire organs as well as the patient. Blood clotting and sludging are common occurrences during and after invasive surgery; thus, it is imperative to find safe procedures to reduce or prevent these deadly phenomena. All anticoagulants used today, for clot prevention and dissolution, can cause excessive bleeding that can lead to enormous medical expense to provide control, otherwise causing patient death. Protein C is a natural protein and is the pivotal anticoagulant in the blood. Due to the mechanism of converting the zymogen protein C (ZPC) to active protein C (APC), only when and where it is needed, and their respective half-lives in the body, the natural anticoagulant, antithrombotic characteristics of APC can be utilized without causing bleeds.

Gelatinase B/matrix metalloproteinase-9 contributes to cellular infiltration in a murine model of zymosan peritonitis.

Murine zymosan-induced peritonitis represents a well-defined model of acute inflammation. However, the molecular mechanisms by which leukocytes degrade basement membranes during extravasation into the peritoneum are not clear. Gelatinase B (MMP-9) is thought to participate in cellular migration, yet its role in leukocyte transmigration through endothelia during inflammation remains controversial. The aim of the present study was to evaluate the role of MMP-9 in the cell influx during zymosan-induced experimental peritonitis. In zymosan-treated Balb/c mice MMP-9 and its natural inhibitor (tissue inhibitor of metalloproteinase 1 - TIMP-1) were present in the peritoneal fluid and plasma at the time of peritoneal neutrophil (polymorphonuclear leukocyte - PMN) infiltration and persisted there until the time of monocytes/macrophages influx. To probe the function of gelatinases, gelatinase B-deficient mice (MMP-9(-/-)) were used as well as Balb/c mice treated with cyclic CTTHWGFTLC (INH), a specific peptide inhibitor of gelatinases. The studies revealed that in either group of mice deprived of MMP-9 activity, PMN infiltration was impaired at the time of their maximal extravasation (6h) while tumor necrosis factor alpha (TNF-alpha), cytokine-induced neutrophil chemoattractant (KC) and interleukin 10 (IL-10) levels were not changed. At later stages (24 h post-zymosan) a significant increase in PMNs was observed in MMP-9(-/-) mice, but not in the inhibitor-treated mice, in comparison to their respective controls. Moreover, intraperitoneal (i.p.) injection of recombinant mouse pro-MMP-9 induced leukocyte accumulation in peritoneum. Collectively, the findings indicate that gelatinase B participates in leukocyte transmigration; however, its function can be compensated by other mechanisms.

Modified apoptotic molecule (BID) reduces hepatitis C virus infection in mice with chimeric human livers.

Hepatitis C virus (HCV) encodes a polyprotein consisting of core, envelope (E1, E2, p7), and nonstructural polypeptides (NS2, NS3, NS4A, NS4B, NS5A, NS5B). The serine protease (NS3/NS4A), helicase (NS3), and polymerase (NS5B) constitute valid targets for antiviral therapy. We engineered BH3 interacting domain death agonist (BID), an apoptosis-inducing molecule, to contain a specific cleavage site recognized by the NS3/NS4A protease. Cleavage of the BID precursor molecule by the viral protease activated downstream apoptotic molecules of the mitochondrial pathway and triggered cell death. We extended this concept to cells transfected with an infectious HCV genome, hepatocytes containing HCV replicons, a Sindbis virus model for HCV, and finally HCV-infected mice with chimeric human livers. Infected mice injected with an adenovirus vector expressing modified BID exhibited HCV-dependent apoptosis in the human liver xenograft and considerable declines in serum HCV titers.

Effects of human prorenin in rats transgenic for human angiotensinogen.

The physiological role of prorenin is unknown; however, the possibility that prorenin inhibits renin locally has been suggested. We tested the hypothesis that prorenin may be an endogenous competitor for renin uptake in the tissue. We also investigated whether prorenin can be activated to active renin and affect mean arterial pressure (MAP). Isolated perfused hindquarters of rats transgenic for human angiotensinogen were infused with human renin and/or prorenin. The plateau phase of angiotensin (Ang) I release 15 minutes after cessation of infusions was used as a parameter for renin uptake. Renin (10 ng/mL for 15 minutes) caused sustained release of Ang I (153+/-16 fmol/mL). Coinfusion with a 15-fold excess of prorenin did not affect local Ang I formation (153+/-19 fmol/mL). Prorenin infusion alone showed no activation to active renin. In addition, we investigated MAP and plasma Ang II levels after injection of saline (DeltaMAP, -1+/-2 mm Hg; 40+/-5 fmol/mL Ang II), 9 ng renin (DeltaMAP, +37+/-3 mm Hg; 378+/-39 fmol/mL), and 144 ng prorenin (DeltaMAP, +10+/-5 mm Hg; 61+/-5 fmol/mL) and the coinjection of renin and prorenin (DeltaMAP, +41+/-4 mm Hg; 305+/-23 fmol/mL) in anesthetized rats. The data show that prorenin was not activated to active renin and did not affect MAP in short-term experiments. Renin-induced Ang formation was not affected by prorenin. Renin may have been taken up specifically because of its physical and chemical properties or because of nonspecific sequestration in the extravascular space. We conclude that prorenin does not act as an endogenous antagonist for the long-lasting effects of renin in the vascular wall. Moreover, prorenin does not affect acute renin-related effects on blood pressure.

Effect of leader peptides on the permeability of mitochondria.

Peptides with sequences based on the leader sequence of yeast cytochrome c oxidase subunit IV (pCOX IV-(1-25)) activate the electrophoretic uptake of K+ and other cations such as tetraethylammonium and lysine by rat liver mitochondria with EC50 = 11-15 microM. Uptake of these cations is dependent on respiration and is prevented by uncoupling agents, and the Vmax for K+ is 1.2-1.5 micromol/min/mg. Albeit more slowly, the non-electrolytes mannitol and sucrose are also transported by this pathway. Treatment of the peptides with proteinase K eliminates the stimulatory effect. Since the stimulated rate is not inhibited by ATP or by cyclosporin, we conclude that this pathway is not related to the mitochondrial KATP channel or the Ca2+-dependent permeability transition pore. Transport is stimulated by pCOX IV-(1-23), pCOX IV-(1-22), and pCOX IV-(1-12)Y, but not by a 13-amino acid peptide representing the nuclear location sequence of the SV40 large T antigen, which is responsible for directing that protein to the nucleus. Spermine, which has four positive charges, also has no stimulatory effect, and an amphiphilic 22-residue peptide derived from antithrombin III with seven net charges is only one-twentieth as effective as pCOX IV-(1-22). Thus, these data indicate that the sequence/structure is important for activation of transport. We also demonstrate that mitochondrial uncoupling, previously reported to be induced by these peptides, actually reflects coupled accumulation of salt. In view of our findings, it is also likely that the lytic effects attributed to these peptides are secondary to swelling and are not due to membrane damage per se. Finally, we show that, in non-ionic media, the peptide is an inhibitor of cytochrome c oxidase.

[A case of coronary artery embolism after double valve replacement].

A 69-year-old woman with combined valvular heart disease (mitral regurgitation and aortic regurgitation), ascending aortic aneurysm, and atrial fibrillation underwent double valve replacement (DVR) and, ascending aortic wall plication. The postoperative thrombo-test level was around 20%. The ST elevation on ECG (II, III, aVFm, V4 approximately V6) with chest pain were recognized on the 13 th postoperative day. She was diagnosed as having acute myocardial infarction, and percutaneous transluminal coronary recanalization was performed immediately. The coronary angiogram showed occlusion at the left anterior descending branch (#8). This lesion could be recanalized by 6,000 U plasminogen pro activator (pro-UK) administration. The cineangiogram on the 35th postoperative day, revealed complete recanalization of this occlusion. Several cases of acute myocardial infarction associated with valvular heart diseases has been reported previously in Japan. However, there has been no report, except for this case, demonstrating occlusion in the coronary artery after prosthetic replacement and successful PTCR. So, this case is the first report on that point.

Thrombolysis with prourokinase versus urokinase: an in vitro comparison.

PURPOSE: Despite advantages demonstrated in vitro, no single thrombolytic agent has been clearly shown to be superior to another in the clinical setting. Prourokinase has recently received attention as a new thrombolytic agent with higher fibrin specificity. The thrombolytic activity of prourokinase, however, remains ill defined. The purpose of this study was to evaluate thrombolysis with prourokinase in comparison to urokinase in vitro. METHODS: We used an in vitro parallel channel perfusion model that simulates catheter-directed thrombolysis in the peripheral arterial system. Radiolabeled thrombi were subjected to 90 minutes of endhole catheter-directed infusion with either prourokinase 5000 IU/ml, urokinase 5000 IU/ml; or 5% dextrose in water at 4 ml/hr. RESULTS: Prourokinase and urokinase were found to be equivalent with respect to thrombolytic effect. Percent lysis was maximal at 90 minutes in both the urokinase and prourokinase groups. Prourokinase and urokinase were found to be equally effective in restoring flow through thrombosed graft segments. CONCLUSION: Prourokinase appears to offer little benefit over urokinase with respect to thrombolytic activity in an in vitro model that closely resembles the clinical setting. If prourokinase is to be accepted as an alternative to urokinase, advantages must relate to differences in fibrin specificity.

[Pro-cathepsin D can activate in vitro pro-cathepsin B secreted by ovarian cancers]. / La pro-cathepsine D peut activer in vitro la pro-cathepsine B sécrétée par les cancers ovariens.

A precursor form of cathepsin B (Mr 45-47 kd) was purified from ascitic fluids of patients with ovarian adenocarcinomas. Following pepsin activation, this precursor produced a 33 kd cathepsin B-like proteinase closely related to lysosomal cathepsin B. A similar activation was found using the 52 kd pro-cathepsin D secreted by the MCF7 human breast cancer cells. This activation was a time, dose and pH dependent process. These results suggest that the 52 kd pro-cathepsin D may be involved in the early steps of the "metastatic cascade", activating pro-cathepsin B in an acidic environment.