Comparative hydrolysis and plasma protein binding of cis-platin and carboplatin in human plasma in vitro

Platinum-based anti-cancer drugs are widely used to treat cancer in patients, but they also exhibit severe toxic side-effects. Considering that cis-platin and carboplatin are intravenously administered, their biotransformations in the bloodstream are likely to be directly involved in determining their toxic side-effects, but they are poorly understood. We added pharmacologically relevant doses of cis-platin or carboplatin to human plasma from healthy male or female volunteers in vitro at 37 °C and determined the platinum-distribution in plasma after 5 min, 3 h and 24 h using size exclusion chromatography-inductively coupled plasma atomic emission spectrometry (SEC-ICP-AES). The results revealed a negligible inter-individual variation of the platinum-distribution between males and females and faster hydrolysis of cis-platin than carboplatin. Related to this, 95% of platinum was protein-bound 24 h after the addition of cis-platin to plasma, whereas 40% of platinum was protein-bound in the case of carboplatin. Interestingly, cis-platin and carboplatin-derived platinum species appeared to bind to the same 3 plasma proteins at the 3 h time point and thereafter. The analysis of cis-platin and carboplatin-spiked phosphate buffered saline (PBS) revealed a common platinum-containing hydrolysis product that was also detected in plasma. Since cis-platin is associated with more toxic side-effects in patients than carboplatin (even though it is administered at lower doses), our in vitro data suggest that the toxic side-effects of the investigated platinum-drugs may be predominantly determined by the indiscriminate translocation of the parent drugs to malignant and healthy cells. This information may help to mitigate the toxic side-effects of platinum-containing drugs by devising strategies to delay the influx of the parent drugs into non-target tissues.     

Alleviation of genotoxic effects of cyclophosphamide using encapsulation into liposomes in the absence or presence of vitamin C

Cyclophosphamide (CP) is a widely used anticancer and immunosuppressant that induces oxidative stress. To ameliorate the side effects resulted from CP treatment, liposomes were tested as an efficient drug delivery system with or without vitamin C as an antioxidant. CP resulted in clastogenic and cytotoxic effects that significantly increased for the total chromosomal aberrations as well as the numerical ones in the CP group (150.8 and 6, respectively) than the control group (6.6 and 0.0) as mean values at p < 0.05. Micronucleus assay showed a significant increased micronucleated polychromatic erythrocytes percentage (MNPCEs% = 11.7%) and a significant decrease of polychromatic to normochromatic erythrocytes ratio (0.551) when compared to the group treated with liposomised CP and vitamin C (3.44%; 0.795, respectively) at p < 0.05. Also, the total glutathione S-transferase activity as a body antioxidant enzyme was decreased from 52.2 in the control to 16.09 nmol/min/mg protein in CP group at p < 0.05, while the highly significant amelioration results were observed in the liposomised vitamin C and CP group (40.88 nmol/min/mg protein). Our findings support the potential use of CP in a liposomal formulation doped with vitamin C to diminish the potential side effects of the agent.     

Variations in plasma motilin, somatostatin, and pancreatic polypeptide concentrations and the interdigestive myoelectric complex in dog

We have looked at the plasma concentrations of motilin, pancreatic polypeptide (PP), and somatostatin (STS) during the various phases of the interdigestive motor complex (IDMC) in dogs. As expected, motilin cyclical increase was always associated with the phase III of the IDMC. Statistical analysis of PP variations revealed a significant rise 10 min before duodenal phase III; however, in individual animals, this relationship was inconsistent. Although a dose-related increase in PP blood levels was induced by administration of synthetic canine motilin (0-200 ng kg-1 iv), fasting plasma levels of PP were not correlated with the concentrations of circulating endogenous motilin. After truncal vagotomy, while motilin release and the intestinal motility pattern remained unaltered, the phase III associated cyclical increases of PP disappeared. Infusion of physiological amounts of PP (1 microgram kg-1 h-1 for 3 h) mimicking the postprandial release failed to reproduce a fed pattern type of intestinal motility and of motilin secretion. No statistical correlation could be established between STS plasma levels and the motor activity of the intestine. STS plasma levels were not correlated with circulating concentrations of motilin and the exogenous administration of physiological doses of synthetic canine motilin failed to modify STS plasma levels. Morphine (200 micrograms kg-1 iv) stimulated only the release of motilin. These data suggest that the role played by circulating concentrations of PP and STS in the control of the IDMC in dog is at most minimal.     

A novel copper complex induces paraptosis in colon cancer cells via the activation of ER stress signalling

Platinum anticancer drugs have been used for three decades despite their serious side effects and the emerging of resistance phenomena. Recently, a phosphine copper(I) complex, [Cu(thp)(4)][PF(6)] (CP), gained special attention because of its strong antiproliferative effects. CP killed human colon cancer cells more efficiently than cisplatin and oxaliplatin and it overcame platinum drug resistance. CP preferentially reduced cancer cell viability whereas non-tumour cells were poorly affected. Colon cancer cells died via a programmed cell death whose transduction pathways were characterized by the absence of hallmarks of apoptosis. The inhibition of 26S proteasome activities induced by CP caused intracellular accumulation of polyubiquitinated proteins and the functional suppression of the ubiquitin-proteasome pathway thus triggering endoplasmic reticulum stress. These data, providing a mechanistic characterization of CP-induced cancer cell death, shed light on the signaling pathways involved in paraptosis thus offering a new tool to overcome apoptosis-resistance in colon cancer cells.     

Separation of rat serum antisuppressor antibodies eliminating T-suppressors and stimulating their generation in mice in vivo

Immunization of rats with enriched murine specific T-suppressors (STS) permitted obtaining the antisuppressor serum (ASS) which selectively inactivated in vitro the capacity of the STS to inhibit the proliferation of T-lymphocytes in a mixed lymphocyte culture in response to allo-antigens. Two opposite effects of the ASS in vivo were demonstrated: elimination of T-suppressors (on ASS administration 4 days after immunization) and stimulation of their formation (on ASS administration before immunization or to non-immunized mice). It is assumed that the two opposite effects of the ASS in vivo are caused by two different types of antibodies to unidentified markers of STS to an antigen of differentiated STS and to an antigen expressed on their precursors.     

Ion-pair reversed-phase HPLC: assay validation of sodium tanshinone IIA sulfonate in mouse plasma

Sodium tanshinone IIA sulfonate (STS), a hydrophilic ionic substance, is used as a cardiovascular drug. An ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) method for the determination of STS in mouse plasma was initially developed. The assay involved a rapid and simple extraction process and subsequent detection at 271 nm. The retention time for STS was 7.5 min. Based on extracted STS standard mouse plasma at 1.5,10 and 50 microg/ml, the assay precision were 2.7, 2.1 and 1.7% with a mean accuracy of 96.7, 98.5 and 99.4%, respectively. At plasma concentration of 1.5, 50 and 75 microg/ml, the mean recovery of STS were 93.1, 96.3 and 97.5%. The limit of detection (LOD) and limit of quantification (LOQ) for STS was 0.1 microg/ml and 0.5 microg/ml, respectively. Linear responses were observed over a wide concentration range (0.5-100 microg/ml) for STS in mouse plasma. STS can be detected after intravenous administration. This method was performed for the first time in pharmacokinetic studies of STS in the mouse.     

Density functional theory and surface enhanced Raman spectroscopy characterization of novel platinum drugs

There is considerable interest in the development of novel platinum-based anticancer drugs that overcome the disadvantages associated with the widely used drug cisplatin, which are its inactivity against some types of tumors and its toxic side effects. In this study we show the suitability of normal Raman spectroscopy (NRS) and surface enhanced Raman spectroscopy (SERS), assisted by density functional theoretical (DFT) calculations, for the characterization of Pt complexes. The Pt complexes studied include the established drugs cisplatin and carboplatin, as well as five novel Pt complexes with anticancer activity. DFT calculations at the B3LYP/LanL2DZ level are a good prediction of the experimental NRS spectra of small and medium sized Pt complexes. The use of SERS allows the investigation of Pt complexes at physiological concentrations, and the binding strengths of the different ligands can be determined. The formation of positively charged hydrolysis products may be necessary for SERS activity. The exiting group in the hydrolysis reaction can be identified.     

Coelomic pouch formation in reconstructing embryos of the starfish Asterina pectinifera

The morphogenetic processes of coelomic pouch (CP) formation in starfish embryos that were experimentally dissociated and induced to undergo reconstruction were studied. An analysis of these embryos randomly chosen from several cultures showed that CP always form on either side of the esophagus, even though the CP formation can differ in timing of initiation and duration, and can vary in number and size from embryo to embryo. Successive observations of CP formation in living embryos revealed two distinct sequences of CP development that were accompanied by different appearances of the blastocoele. These processes were named 'enterocoelic-like' and 'schizocoelic-like' CP formation. The former resembled normal development and occurred in embryos with a transparent blastocoele. The latter was characterized by the aggregation and epithelialization of mesenchyme-like cells on either side of the esophagus and was observed in embryos possessing a cloudy blastocoele. In a few embryos, both types of CP formation were seen in the same individual ('mosaic type' CP formation). Thick sections of embryos possessing a cloudy blastocoele revealed that aggregates of mesenchyme-like cells undergoing CP formation directly contact the developing esophagus. Together, these data demonstrate flexibility in the morphogenetic processes that regulate CP formation, and suggest that positional cues in the esophagus regulate the placement of CP.     

Recent developments in ruthenium anticancer drugs

Interest in Ru anticancer drugs has been growing rapidly since NAMI-A ((ImH(+))[Ru(III)Cl(4)(Im)(S-dmso)], where Im = imidazole and S-dmso = S-bound dimethylsulfoxide) or KP1019 ((IndH(+))[Ru(III)Cl(4)(Ind)(2)], where Ind = indazole) have successfully completed phase I clinical trials and an array of other Ru complexes have shown promise for future development. Herein, the recent literature is reviewed critically to ascertain likely mechanisms of action of Ru-based anticancer drugs, with the emphasis on their reactions with biological media. The most likely interactions of Ru complexes are with: (i) albumin and transferrin in blood plasma, the former serving as a Ru depot, and the latter possibly providing active transport of Ru into cells; (ii) collagens of the extracellular matrix and actins on the cell surface, which are likely to be involved in the specific anti-metastatic action of Ru complexes; (iii) regulatory enzymes within the cell membrane and/or in the cytoplasm; and (iv) DNA in the cell nucleus. Some types of Ru complexes can also promote the intracellular formation of free radical species, either through irradiation (photodynamic therapy), or through reactions with cellular reductants. The metabolic pathways involve competition among reduction, aquation, and hydrolysis in the extracellular medium; binding to transport proteins, the extracellular matrix, and cell-surface biomolecules; and diffusion into cells; with the extent to which individual drugs participate in various steps along these pathways being crucial factors in determining whether they are mainly anti-metastatic or cytotoxic. This diversity of modes of action of Ru anticancer drugs is also likely to enhance their anticancer activities and to reduce the potential for them to develop tumour resistance. New approaches to metabolic studies, such as X-ray absorption spectroscopy and X-ray fluorescence microscopy, are required to provide further mechanistic insights, which could lead to the rational design of improved Ru anticancer drugs.     

Sensitive gas chromatographic determination of the cyclophosphamide metabolite 2-dechloroethylcyclophosphamide in human plasma

Cyclophosphamide (CP) is one of the most frequently used anticancer agents. It is a prodrug requiring activation before exerting cytotoxicity. CP is deactivated to 2-dechloroethylcyclophosphamide (2-DCECP) with formation of an equimolar amount of chloroacetaldehyde. The aim of this study was to develop and validate a sensitive and simple assay for 2-DCECP in plasma of patients treated with CP. Sample pre-treatment consisted of solid-phase extraction of 500 μl of plasma over OASIS HLB (1 ml) cartridges with trofosfamide as internal standard. Separation and detection of underivatized 2-DCECP was performed with capillary gas chromatography with nitrogen/phosphorous selective detection. Extraction recovery of 2-DCECP exceeded 87%. No interference from endogenous compounds, other metabolites of CP and frequently co-administered drugs was detected. The assay was linear in the range of 5–5000 ng/ml in plasma. Accuracy, within-day and between-day precision were less than 11% for the complete concentration range. In plasma, 2-DCECP was stable for at least 1 month when kept at −70°C. Analysis of samples from patients treated with CP demonstrated the applicability of the assay. In conclusion, a sensitive and simple assay for 2-DCECP in plasma, which meets the current requirements for bioanalytical assays, was developed.     

Prior exposure to indole-3-carbinol decreases the incidence of specific cyclophosphamide-induced developmental defects in mice

BACKGROUND: Indole-3-carbinol (I3C) is a product of the hydrolysis of glucobrassicin that is found in cruciferous vegetables. I3C can intervene in toxic processes that are mediated by oxidative mechanisms because it possesses the chemical and pharmacokinetic properties necessary to provide a free radical trap. Cyclophosphamide (CP) is a bifunctional alkylating agent known to produce DNA damage and to cause developmental toxicity, including malformations, in laboratory animals. METHODS: Pregnant CD-1 mice were given a 100 mg/kg dose of I3C 24 or 48 hr before administration of 20 mg/kg CP on gestation day 10 (GD 10). Controls were given the vehicle (DMSO), I3C, or CP. This regimen was carried out to determine if I3C could protect against the developmental toxicity of alkylating agents, such as CP. Dams were sacrificed on GD 17 and their litters were examined for adverse effects. RESULTS: Treatment with I3C 48 hr before CP administration was associated with decreased fetal limb and tail malformations. Limb malformation incidences were reduced from 42% litters affected in the CP control to 16% in the I3C/CP 48-hr treatment group, and tail malformations were reduced from 45% in the CP control to 16% in the I3C/CP 48-hr treatment group, indicating a protective effect of prior exposure to I3C. I3C given 24 hr before CP had no significant protective effect, while having an apparently adverse consequence with regard to the incidence of talipes. CONCLUSIONS: Exposure of a developing mammal to indole-3-carbinol before exposure to cyclophosphamide during organogenesis can influence the teratogenicity of cyclophosphamide.     

Evidence for acrolein-modified DNA in peripheral blood leukocytes of cancer patients treated with cyclophosphamide

Monitoring human populations for specific DNA modifications has been made possible by developing highly sensitive immunoassays employing antibodies specific for carcinogen-DNA adducts. While these techniques have been used to follow occupationally and environmentally exposed populations, results have been limited by the lack of exposure data with which to correlate adduct formation. Cancer patients treated with precisely known doses of anticancer drugs can be studied to examine the association between drug dose and adduct formation. This study examined acrolein-modified DNA in patients treated with the anticancer drug cyclophosphamide (CP) and in newly diagnosed patients prior to treatment. Employing 2 different detection methods, enzyme-linked immunosorbent assay (ELISA) and immuno-dot blot (IDB), acrolein-modified DNA was identified in a total of 6 of 12 (50%) treated patients and in 0 of 15 untreated patients. Formation of acrolein-modified DNA was examined as a function of lifetime CP dose, recent CP dose, time since last treatment, regime of treatment, and smoking history; however no clear trends were observed.     

Role of steroid sulfatase in local formation of estrogen in post-menopausal breast cancer patients

More than two-thirds of breast cancers occur in post-menopausal women, and depend on the estrogens for their proliferation and survival. For the treatment of estrogen-dependent breast cancers, two major treatment options are now available. One is selective estrogen receptor modulator (SERM) such as Tamoxifen and another is aromatase inhibitor such as Anastrozole, Letrozole and Exemestane, which reduce local in situ formation of estrogens. Although these therapies are clinically active for advanced and early breast cancers, de novo and/or acquired resistance to SERM and/or aromatase inhibitors are also clinical problem. Recent studies suggest that local formation of estrogens in the breast tumors is more important than circulating estrogen in plasma for the growth and survival of estrogen-dependent breast cancer in post-menopausal women. The rationale for the importance of local formation of estrogens is based on the following evidences. Estradiol (E2) levels in breast tumors are equivalent to those of pre-menopausal patients, although plasma E2 levels are 50-fold lower after menopause. E2 concentrations in breast tumors of post-menopausal women are 10–40 times higher than serum level. Biosynthesis of estrogens in breast tumors tissues occurs via two major different routes, one is aromatase pathway and another is steroid-sulfatase (STS) pathway. Whereas many studies has been reported about aromatase inhibitor and its clinical trial results in breast cancer patients, limited information are available regarding to other estrogen regulating enzymes including STS, its role in breast tumors and STS inhibitors. STS is the enzyme that hydrolyses estrone 3-sulfate (E₁S) and dehydroepiandrosterone-sulfate (DHEA-S) to their active un-sulfoconjugated forms, thereby stimulating the growth and survival of estrogen-dependent breast tumors. It has been well known that E₁S level are much higher than E2 level both in plasma and tumor of post-menopausal patients. Recent reports show that more than 80% of breast tumors are stained with anti-STS antibody and the expression of STS is an independent prognostic factor in breast cancer. Taking these findings into consideration, local formation of estrogens could be partially synthesized from large amount of E₁S by STS, which exist in breast cancer. On the other hand, aromatase localizes in stroma and adipocyte surrounding breast cancer. Furthermore, since estrogen formation from E₁S and DHEA-S (STS pathway) cannot be blocked by aromatase inhibitors, STS is thought to be a new molecular target for the treatment of estrogen-dependent tumor post-SERM and/or aromatase inhibitors. In this symposium, these recent rationale for the importance of STS in post-menopausal breast cancer patients is reviewed as well as STS inhibitor.     

Properties of streptokinase incorporated into polyethylene glycol microcapsules

Thrombolytic therapy with high doses of streptokinase (SK), which are required due to its rapid clearance from the bloodstream, is accompanied by side effects. In this work, the SK was incorporated into water-soluble polyethylene glycol (PEG) microcapsules with the double emulsification method in order to increase its lifetime in bloodstream and decrease side effects. Four preparations of SK*PEG-microcapsules with a high degree of the SK enclosure (～90–91%) and total retention of fibrinolytic activity were produced under varying emulsification conditions (PEG molecular mass 20 or 40 kDa and PEG/SK ratio 12 or 8 mg of PEG/1000 IU SK). SK was released from the PEG-microcapsules at different rates: the time of complete release varied from 45 to 90 min (pH 7.4, 37°C). Comparative in vitro study of thrombolytic activity and side effects of the SK in a free and encapsulated state was conducted. It was found that the rate of human plasma clot lysis under the action of encapsulated SK preparations is equal (with exception of a short lag-period) to the rate of lysis induced by the free SK, provided that the doses were equal (500 IU/mL). Furthermore, the SK*PEG-microcapsules caused the reduced exhaustion of plasminogen and fibrinogen in plasma when compared with the free SK.     

Clinical application of recombinant human erythropoietin for treatments in patients with head and neck cancer

Summary The therapeutic effects of intravenous recombinant human erythropoietin (r-hEPO) administration on anemia induced by radiation therapy (3 cases), chemotherapy (18 cases) and combined therapies (5 cases) in patients with head and neck malignancies were examined. The effectiveness was evaluated by the changes in the hemoglobin concentration examined before and after the r-hEPO administration. The r-hEPO administration combined with anticancer therapies improved anemia induced by all three treatments. The therapeutic effectiveness of r-hEPO injection was also noted on anemia induced by all of four different chemotherapeutic regimens that have been ordinarily used for head and neck malignancies. Furthermore, the efficacy of the different dose schedules, 3000 IU (12 cases) or 6000 IU (14 cases), three times a week, was compared. Both of the r-hEPO dose schedules were effective for anemia, but the efficacy of 6000 IU was superior to that of 3000 IU. No significant changes were observed in the number of white blood cells and platelets and the results of biochemical examinations after the r-hEPO injection. There were no objective side-effects related to the r-hEPO administration. These results suggest that anemia induced by chemotherapy and/or radiotherapy could be prevented by r-hEPO administration. The addition of r-hEPO to anticancer therapies would make it possible to pursue the planned therapeutic schedules, prevent the decrease of immunity after allogeneic blood transfusion and bring about an improvement in the prognosis of patients with malignancies.     

Protein thiol oxidation by haloperidol results in inhibition of mitochondrial complex I in brain regions: comparison with atypical antipsychotics

Usage of 'typical' but not 'atypical' antipsychotic drugs is associated with severe side effects involving extrapyramidal tract (EPT). Single dose of haloperidol caused selective inhibition of complex I in frontal cortex, striatum and midbrain (41 and 26%, respectively) which was abolished by pretreatment of mice with thiol antioxidants, alpha-lipoic acid and glutathione isopropyl ester, and reversed, in vitro, by disulfide reductant, dithiothreitol. Prolonged administration of haloperidol to mice resulted in complex I loss in frontal cortex, hippocampus, striatum and midbrain, while chronic dosing with clozapine affected only hippocampus and frontal cortex. Risperidone caused complex I loss in frontal cortex, hippocampus and striatum but not in midbrain from which extrapyramidal tract emanates. Inhibition of the electron transport chain component, complex I by haloperidol is mediated through oxidation of essential thiol groups to disulfides, in vivo. Further, loss of complex I in extrapyramidal brain regions by anti-psychotics correlated with their known propensity to generate side-effects involving extra-pyramidal tract.     

2-O-α-D-Glucopyranosyl-l-ascorbic acid as an antitumor agent for infusion therapy

Ascorbic acid (AA) has been reported as a treatment for cancer patients. Intravenous (iv) administration of high-dose AA increases plasma AA levels to pharmacologic concentrations and generates reactive oxygen species (ROS) to exert anti-tumor activity via enhancement of oxidative stress. However, AA is very unstable in aqueous solutions and it is impossible to preserve AA for a long period in a solution. 2-O-α-D-Glucopyranosyl-l-ascorbic acid (AA-2G), which is a glucoside derivative of AA, has been found to exhibit much higher stability than AA in aqueous solutions and it shows vitamin C activity after enzymatic hydrolysis to AA. To evaluate the effectiveness of AA-2G for cancer treatment, we examined the antitumor activity of AA-2G to murine colon carcinoma (colon-26) cells and in tumor-bearing mice. AA-2G did not show cytotoxicity to colon-26 cells, whereas AA exhibited a significant cytotoxic effect in a concentration-dependent manner. In colon-26 tumor-bearing mice, iv administration of high-dose AA-2G as well as that of AA significantly inhibited tumor growth. Experiments on the biodistribution and clearance of AA-2G in tumor-bearing mice showed that AA-2G was rapidly hydrolyzed to AA and exhibited significant antitumor activity. Treatment of tumor-bearing mice with AA-2G tended to increase plasma malondialdehyde level. These results indicated that the antitumor activity of AA-2G was caused by ROS generated by AA released by rapid hydrolysis of AA-2G.     

Glycosyl and polyalcoholic prodrugs of lonidamine

Polyhydric alcohol derivatives of the anticancer agent lonidamine (LND) have been synthesized. The increased water solubility showed by prodrugs 4, 7, and 25 together with their logP values (2.19, 2.55, and 2.54, respectively) and chemical stability might be beneficial for prodrugs absorption after oral administration. Moreover, the new prodrugs undergo enzymatic hydrolysis in plasma and release LND demonstrating that they are promising candidates for in vivo investigations.     

Effects of morphine and opioid peptides on plasma levels of atrial natriuretic peptide

The effect of opiate ligand administration on plasma levels of atrial natriuretic peptide (ANP) was studied in awake, freely moving Sprague-Dawley rats. Prior to and following the intracerebroventricular (icv) or central venous (iv) injection of morphine (MS), leu-enkephalin (Leu-enk), dynorphin (Dyn) or beta-endorphin (B-endor), plasma samples were obtained for measurement of ANP concentrations by radioimmunoassay. MS was 10 times more potent when given icv than when given iv to increase plasma ANP levels. Icv injection of Leu-enk decreased plasma ANP concentrations. Dyn and B-endor administration (iv or icv) did not alter the plasma concentration of ANP. These effects of MS and Leu-enk on plasma concentrations of ANP appear to be mediated through actions on the central nervous system. MS, Leu-enk, B-endor, and Dyn given icv, produced elevations of plasma norepinephrine (NE) and epinephrine (Epi) concentrations. When MS was given icv, mean Epi and NE plasma levels increased 10-50 times the increases noted with B-endor, Leu-enk and Dyn. A role of catecholamines in mediating MS-stimulated ANP release is supported by the observation that ganglionic blockade with chlorisondamine significantly attenuated the increase of plasma ANP levels. MS, but not B-endor, Leu-enk and Dyn, acts within the brain to increase plasma levels of ANP. MS-induced elevations of plasma ANP levels may be dependent on an intact autonomic nervous system.     

Enzymes and inhibitors in leu-enkephalin in metabolism in human plasma

The enzymes degrading leucine enkephalin in human plasma and the inhibitors active on these enzymes were studied by kinetic and chromatographic techniques. Data obtained evidence the existence of complex kinetics of leu-enkephalin hydrolysis and of formation of its hydrolysis byproducts. These appear to originate from the combined effect of further hydrolysis of the enkephalin's fragments after their release and of competition between the different enzymes present in plasma. Chromatographic separation of plasma proteolysis inhibitors indicates the existence of several pools of substances acting on all three enzyme groups that degrade leu-enkephalin. The partial specificity of these substances induces competition effects: consequently, the actual protection over leu-enkephalin is considerably lower that the total inhibitory activity. That notwithstanding, plasma inhibitors control enkephalin hydrolysis to a relevant extent, while they modify only slightly the ratio of hydrolysis between the different enzymes. This latter parameter—and specifically the large prevalence of aminopeptidases over dipeptidylaminopeptidases and dipeptidylcarboxypeptidases—appears controlled mainly by kinetic factors.