Analysis of radiotherapy in 1054 patients with primary central nervous system lymphoma treated from 1985 to 2009.

AIMS: Data on primary central nervous system lymphoma that had been collected through surveys for four consecutive periods between 1985 and 2009 were analysed to evaluate outcomes according to treatment. MATERIALS AND METHODS: All had histologically proven disease and had received radiotherapy. No patients had AIDS. Among 1054 patients, 696 died and 358 were alive or lost to follow-up. The median follow-up period for surviving patients was 37 months. RESULTS: For all patients, the median survival time was 24 months; the 5 year survival rate was 25.8%. Patients treated with methotrexate-based chemotherapy and radiation had a higher 5 year survival rate (43%) than those treated with radiation alone (14%) and those treated with non-methotrexate chemotherapy plus radiation (20%), but differences in relapse-free survival were smaller among the three groups. The 5 year survival rate was 25% for patients treated with whole-brain irradiation and 29% for patients treated with partial-brain irradiation (P = 0.80). Patients receiving a total dose of 40-49.9 Gy had a higher 5 year survival rate (32%) than those receiving other doses (21-25%, P = 0.0004) and patients receiving a whole-brain dose of 30-39.9 Gy had a higher 5 year survival rate (32%) than those receiving ≥40 Gy (13-22%, P < 0.0005). Patients receiving methotrexate-based chemotherapy and partial-brain radiotherapy (≥30 Gy) had a 5 year survival rate of 49%. CONCLUSIONS: The optimal total and whole-brain doses may be in the range of 40-49.9 and <40 Gy, respectively, especially in combination with chemotherapy. Patients receiving partial-brain irradiation had a prognosis similar to that of those receiving whole-brain irradiation. With methotrexate-based chemotherapy, partial-brain radiotherapy may be worth considering for non-elderly patients with a single tumour.

Haemoglobin-vesicles as artificial oxygen carriers: present situation and future visions.

During the long history of development of haemoglobin (Hb)-based O2 carriers (HBOCs), many side effects of Hb molecules have become apparent. They imply the physiological importance of the cellular structure of red blood cells. Hb-vesicles (HbV) are artificial O2 carriers that encapsulate concentrated Hb solution with a thin lipid membrane. We have overcome the intrinsic issues of the suspension of HbV as a molecular assembly, such as stability for storage and in blood circulation, blood compatibility and prompt degradation in the reticuloendothelial system. Animal tests clarified the efficacy of HbV as a transfusion alternative and the possibility for other clinical applications. The results of ongoing HbV research make us confident in advancing further development of HbV, with the expectation of its eventual realization.

Virus inactivation in hemoglobin solution by heat treatment.

To increase the safety of stroma-free hemoglobin solution (SFH) as an artificial oxygen carrier source, we investigated the effect of heat treatment on virus inactivation in hemoglobin solution. The hemoglobin solution spiked with vesicular stomatitis virus (VSV) was treated at 60 degrees C for 1 hr under either an air or CO atmosphere. VSV was inactivated at >5.8 log10 and >6.0 log10 under the air and CO atmosphere, respectively. Although the methemoglobin rate increased after the heat treatment under the air atmosphere, no methemoglobin formation was observed by the treatment under the CO atmosphere. Isoelectric focusing analysis revealed the denaturation of hemoglobin after the heat treatment under the air, while hemoglobin banding was not altered in the carbonylated condition. Some protein bands other than hemoglobin were weakened or disappeared on SDS-PAGE after the heat treatment under both conditions. In addition, the hemoglobin concentration in the SFH was higher after the heat treatment than before the treatment. These findings indicate that the heat treatment under the CO atmosphere inactivates viruses without hemoglobin denaturation, and hence, this method is a promising approach to prepare a safer SFH as artificial oxygen carriers.

[Brain metastases from esophageal cancers: clinical features and treatment results].

Metastatic brain tumors from esophageal cancer are relatively rare. We analyzed the clinical features and results of treatment in 14 cases of brain metastases from esophageal carcinoma. The average time to diagnosis of brain metastases in the 11 patients with metachronous lesions was 13 months. The average age of patients at the diagnosis of brain metastasis was 65 years. Most patients had T4 or N1 disease at the time of diagnosis of esophageal cancer. Performance status of grade 3 was most frequent at the time of diagnosis of brain metastasis. Treatment for brain metastases was surgery followed by radiation in five cases, radiotherapy alone in seven cases, and conservative treatment in two cases. The median survival time of all patients from the treatment of brain metastases was 2 months, with only one patient alive after more than one year. Improvement in neurological symptoms was demonstrated in 42% of cases. These extremely poor treatment results reflect the fact that most patients at the time of diagnosis of brain metastasis had poor performance status and the presence of extracerebral metastases. Therefore, a short-course, high-dose-per-fraction treatment for brain metastases from esophageal cancer should be selected from the viewpoint of quality of life.

Hemoglobin-vesicles as oxygen carriers: influence on phagocytic activity and histopathological changes in reticuloendothelial system.

Hemoglobin-vesicles (HbV) have been developed for use as artificial oxygen carriers (particle diameter, 250 nm) in which a purified Hb solution is encapsulated with a phospholipid bilayer membrane. The influence of HbV on the reticuloendothelial system was studied by carbon clearance measurements and histopathological examination. The HbV suspension ([Hb] = 10 g/dl) was intravenously infused in male Wistar rats at dose rates of 10 and 20 ml/kg, and the phagocytic activity was measured by monitoring the rate of carbon clearance at 8 hours and at 1, 3, 7, and 14 days after infusion. The phagocytic activity transiently decreased one day after infusion by about 40%, but it recovered and was enhanced at 3 days, showing a maximum of about twice the quiescent level at 7 days, and then returned to the normal value at 14 days. The initial transient decreased activity indicates a partly, but not completely, suppressed defensive function of the body. The succeeding increased phagocytic activity corresponds to the increased metabolism of HbV. The histopathological examination with anti-human Hb antibody, hematoxylin/eosin, and oil red O stainings showed that HbV was metabolized within 7 days. Hemosiderin was very slightly confirmed with Berlin blue staining at 3 and 7 days in liver and spleen, though they completely disappeared at 14 days, indicating that the heme metabolism, excretion or recycling of iron proceeded smoothly and iron deposition was minimal. Electron microscopic examination of the spleen and liver tissues clearly demonstrated the particles of HbV with a diameter of about 1/40 of red blood cells in capillaries, and in phagosomes as entrapped in the spleen macrophages and Kupffer cells one day after infusion. The vesicular structure could not be observed at 7 days. Even though the infusion of HbV modified the phagocytic activity for 2 weeks, it does not seem to cause any irreversible damage to the phagocytic organs. These results offer important information for evaluating the safety issues of HbV for clinical use.

Supratentorial astrocytomas and oligodendrogliomas treated in the MRI era.

BACKGROUND: There is at present no consensus on the policy for the treatment of patients with low-grade gliomas (LGGs). METHODS: This report is a retrospective multi-institutional study of 100 patients (ages 16-65 years) with astrocytoma (grade II), oligodendroglioma, anaplastic oligodendroglioma and anaplastic oligoastrocytoma of the supratentorial areas which were treated with surgery and postoperative radiotherapy at five university hospitals in northern Japan between 1990 and 1997 when MRI was routinely used to determine the target volume. Most patients were irradiated with 50-60 Gy. The target volume usually covered the areas with T2 prolongation of MRI with a margin of 2 cm. RESULTS: The disease-specific 5-year survival rate was 87.4% for patients with oligodendroglioma and 75.3% for patients with astrocytoma. Survival for patients with astrocytoma in the MRI era appears to be improved compared with historical controls in the literature. Patients with astrocytoma aged 40 years and under had a significantly better disease-specific survival rate than those over 40 years (P < 0.05) and patients with oligodendroglioma and oligoastrocytoma showed a similar tendency. Patients with astrocytoma who had over 50% of their tumor removed had a significantly better survival rate than those who had less than 50% removed (P < 0.05). Chemotherapy appeared to improve the disease-specific survival rate of patients with oligodendroglioma but not that of patients with astrocytoma. CONCLUSION: Oligodendroglioma has a more protracted course of disease progression than astrocytoma. This particular feature and the sensitivity of LGGs to chemotherapy as well as their relevant prognostic factors, such as age, histopathology and amount of tumor removal, should be taken into account before any decision on treatment methods for LGGs is made.

Photoreduction of autooxidized albumin-heme hybrid in saline solution: revival of its O(2)-binding ability.

Recombinant human serum albumin (rHSA) incorporating 2-[8-[N-(2-methylimidazolyl)]octanoyloxymethyl]-5,10,15,20-tetrakis(alpha,alpha,alpha,alpha-o-pivalamido)phenylporphinatoiron(II)s (Fe(II)Ps) [rHSA-Fe(II)P] is a synthetic hemoprotein which can bind and release O(2) reversibly under physiological conditions (saline solution [NaCl]: 150 mM, pH 7.3) as do hemoglobin and myoglobin. However, the central ferrous ions of Fe(II)Ps are slowly oxidized to O(2)-inactive ferric forms. Based on the UV-vis. absorption spectroscopy, the majority of the autooxidized Fe(III)Ps in albumin are determined to be six-coordinate high-spin complexes with a proximal imidazole and a chloride anion, which show ligand-to-metal charge transfer (LMCT) absorption at 330 nm. Interestingly, photoirradiation of this LMCT band under an argon atmosphere led to reduction of the central ferric iron of Fe(III)P, allowing the revival of the O(2)-binding ability. The ratio of the photoreduction reached a maximum of 83%, which is probably due to the partial dissociation of the axial imidazole. The same photoirradiation under a CO atmosphere provides the corresponding carbonyl rHSA-Fe(II)P. Laser flash photolysis experiments revealed that the reduction was completed within 100 ns. The quantum yields (Phi) of these photoreductions were approximately 0.01.

A nanometer-sized high-spin polyradical: poly(4-phenoxyl-1,2-phenylenevinylene) planarily extended in a non-Kekulé fashion and its magnetic force microscopic images.

A pi-conjugated, but non-Kekulé- and nondisjoint-type poly(1,2-phenylenevinylene) network bearing 4-substituted di-tert-butylphenoxyls was synthesized through a one-pot polycondensation of the star-shaped subpart and the subsequent oxidation, which was persistent even at room temperature. The polyphenoxyl radical with a spin concentration of 0.4 displayed an average S of 10/2. The polyradical with the molecular weight of 3.2 x 10(4) gave a disklike image of ca. 35 x 0.6 nm with both an atomic and a magnetic force microscopy: the molecular image was examined as a nanoscale and single-molecular-based magnetic dot.

Effects of poly(ethyleneglycol)-modified hemoglobin vesicles on agonist-induced platelet aggregation and RANTES release in vitro.

We studied the effects of hemoglobin-vesicles modified with PEG (PEG-HbV), a type of liposome-encapsulated hemoglobin (LEH), on human platelet functions in vitro. The effect of a low concentration of PEG-HbV (Hb; 5.8 mg/dl) was assessed by examining an agonist-induced aggregation response, and that of relatively high concentrations of PEG-HbV (Hb; 0.29, 1 and 2 g/dl) by measuring the release of RANTES (Regulated upon activation, normal T-cell expressed and presumably secreted) from platelets, which is regarded as a marker of platelet activation. The preincubation of platelets with PEG-HbV at 5.8 mg/dl of Hb did not affect platelet aggregation induced by collagen, thrombin and ristocetin. The pretreatment of platelet-rich plasma (PRP) with PEG-HbV at concen trations up to 2 g/dl of Hb had no aberrant effects on the collagen-induced RANTES release. Furthermore, the collagen-induced release of RANTES from PRP was not affected by longer incubation with PEG-HbV at 2 g/dl of Hb. The basal levels of RANTES from PRP were unchanged in the presence of PEG-HbV. These results suggest that PEG-HbV, at the concentrations studied, have no aberrant effects on platelet functions in the presence of plasma.

Electrochemical and ferromagnetic couplings in 4,4',4' '-(1,3,5-benzenetriyl)tris(phenoxyl) radical formation.

4,4',4' '-(1,3,5-Benzenetriyl)tris(2,6-di-tert-butylphenol) was prepared by the cross-coupling of 1,3,5-tribromobenzene and [4-(trimethylsiloxy)phenyl]magnesium bromide. X-ray analysis of the single crystal showed a propeller-like structure with a mean dihedral angle of 39 degrees between the hydroxyphenyl and the core benzene. The phenoxyl mono-, di-, and triradicals were generated by the electrochemical oxidation of the trianion. A stepwise radical formation was revealed by a differential pulse voltammogram, electrolytic ESR spectroscopy, and a comproportionation reaction between the radicals, which was discussed as an effect of the pi-conjugated but non-Kekulé-type coupler. The quartet and triplet ground state for the tri- and diradical, respectively, were confirmed by a SQUID measurement.

Carbon monoxide from heme catabolism protects against hepatobiliary dysfunction in endotoxin-treated rat liver.

BACKGROUND AND AIMS: Liver is a major organ for heme detoxification under disease conditions, but its self-protective mechanisms against the toxicity are unknown. This study aimed to examine roles of carbon monoxide (CO), the gaseous product of heme oxygenase (HO), in ameliorating hepatobiliary dysfunction during catabolism of heme molecules in endotoxemic livers. METHODS: Vascular resistance and biliary flux of bilirubin-IXalpha, an index of HO-derived CO generation, were monitored in perfused livers of endotoxemic rats. Livers were perfused with HbO(2), which captures nitric oxide (NO) and CO, or metHb, a reagent trapping NO but not CO. RESULTS: In endotoxin-pretreated livers where inducible NO synthase and HO-1 overproduced NO and CO, HbO(2) caused marked vasoconstriction and cholestasis. These changes were not reproduced by the NO synthase inhibitor aminoguanidine alone, but by coadministration of zinc protoporphyrin-IX, an HO inhibitor. CO supplementation attenuated the events caused by aminoguanidine plus zinc protoporphyrin-IX, suggesting that simultaneous elimination of these vasorelaxing gases accounts for a mechanism for HbO(2)-induced changes. This concept was supported by observation that metHb did not cause any cholestasis; the reagent captures NO but triggers CO overproduction through rapid degradation of the heme by HO-1. CONCLUSIONS: These results suggest protective roles of CO against hepatobiliary dysfunction caused by heme overloading under stress conditions.

Reaction of nitric oxide with synthetic hemoprotein, human serum albumin incorporating tetraphenylporphinatoiron(II) derivatives.

The reaction of nitric oxide (NO) with a synthetic hemoprotein, the recombinant human serum albumin (rHSA) incorporating eight tetraphenylporphinatoiron(II) derivatives bearing a covalently linked axial base (FeP) [rHSA-FeP], has been investigated. The UV--vis absorption spectrum of the phosphate buffer solution (pH 7.3) of rHSA-FeP showed maxima at 425 and 546 nm upon the addition of NO. The carbonyl rHSA-FeP, in which FePs are six-coordinate CO-adducts, also moved to the same species after bubbling with NO gas. ESR spectroscopy revealed that the incorporated FePs in the albumin formed six-coordinate nitrosyl complexes; the proximal imidazole moiety does not dissociate from the central iron when NO binds to the trans side. The NO-binding affinity of rHSA-FeP (P(1/2)(NO), 1.7 x 10(-6) Torr, pH 7.3, 298 K) was significantly lower than that of FeP itself (P(1/2)(NO), 1.8 x 10(-8) Torr in toluene). Kinetically, this arises from the decreased association rate constant (k(on)(NO), 8.9 x 10(8) M(-1) s(-1) --> 1.5 x 10(7) M(-1) s(-1)). Since NO-association is diffusion controlled, incorporation of the synthetic heme into the albumin matrix appears to restrict the NO access to the central iron(II).

Effects of poly(ethyleneglycol)-modified hemoglobin vesicles on N-formyl-methionyl-leucyl-phenylalanine-induced responses of polymorphonuclear neutrophils in vitro.

[Poly(ethyleneglycol)]-modified hemoglobin vesicles (PEG-HbV), a type of encapsulated hemoglobin, have been developed as artificial oxygen carriers and it is important to evaluate their blood compatibility. We studied the effects of PEG-HbV on human polymor phonuclear neutrophils (PMNs) in vitro, focusing on the functional responses to N-formyl-methionyl-leucyl-phenylalanine (fMLP) as an agonist. The pretreatment of the PMNs with PEG-HbV up to a concentration of 60 mg/dl Hb did not affect the fMLP-triggered chemotactic activity. In parallel to these results, the fMLP-induced upregulation of CD11b (Mac-1) levels on the PEG-HbV-pretreated PMNs was comparable to that of untreated cells. Furthermore, the pretreatment of the PMNs with the PEG-HbV even at 600 mg/dl Hb did not affect the gelatinase B (Matrix methalloproteinase-9 (MMP-9)) release, suggesting that the fMLP-induced release of secondary and tertiary granules was normal. In addition, the fMLP-triggered superoxide production of the PMNs was unchanged by the pretreatment with the PEG-HbV at 600 mg/dl Hb. Thus, these results suggest that PEG-HbV, at the concentrations studied, have no aberrant effects on the fMLP-triggered functions of human PMNs.

Fibrinogen-conjugated albumin polymers and their interaction with platelets under flow conditions.

Albumin polymers, having an average diameter of 1020 +/- 250 nm, were prepared by the disulfide polymerization of recombinant human serum albumin (rHSA) by controlling of the pH and temperature. Fibrinogen could be conjugated on the surface of an albumin polymer using N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). Under flow conditions, the fibrinogen-conjugated albumin polymers (fibrinogen-albumin polymers) were irreversibly attached to the platelet-immobilized surface in the reconstituted blood at a low platelet concentration ([platelet] = 5.0 x 10(4)/microL, a 5-fold diluted platelet concentration), and the attachment was suppressed by the addition of anti-GPIIb/IIIa monoclonal antibodies. It was confirmed that fibrinogen-albumin polymers specifically interacted with GPIIb/IIIa expressed on the surface of the activated platelets. Although platelets with a low platelet concentration were hardly attached to the platelet-immobilized surface under the flow conditions, the addition of fibrinogen-albumin polymers enhanced the attachment of the remaining platelets to the surface, indicating that the fibrinogen-albumin polymers would help the hemostatic ability of platelets at the site of vascular injury of patients in thrombocytopenia.

Kinetics of CO and O(2) binding to human serum albumin-heme hybrid.

The kinetics of the CO and O(2) binding to the synthetic hemoprotein, recombinant human serum albumin (rHSA) incorporating eight 2-[8-¿N-(2-methylimidazolyl)¿octanoyloxymethyl]-5,10,15, 20-tetrakis(o-pivalamido)phenylporphinatoiron(II)s (FePs) [rHSA-FeP(8)] have been investigated by laser flash photolysis. Time dependence of the absorption change accompanied the CO rebinding to rHSA-FeP(8) was composed of three phases. The fastest component was the axial base elimination, and the long-lived biphasic decay corresponds to the direct recombination of CO to the five-N-coordinated FePs in rHSA. The rate constants of the fast and slow phases of the CO association [(fast), (slow)] were determined to be 4.9 x 10(6) M(-)(1) s(-)(1) and 6.7 x 10(5) M(-)(1) s(-)(1), respectively. The initial amplitude after the laser pulse gave the concentration ratio of the fast and slow phases (n = 3); (i) two of the eight FePs exhibited the slow rate constants and (ii) they are presumably accommodated in the second and fifth binding sites of FeP in the albumin structure. The absorption decay following the O(2) photodissociation of rHSA-FeP(8) also showed the same behavior. Thermodynamically, the large DeltaG() of the slow phase of the CO rebinding, which mainly comes from the enthalpic factor, suggests the appearance of additional steric hindrance on the central metal iron of FeP. Furthermore, orientation of the porphyrin plane in rHSA was predicted by molecular simulation, which supports the experimental data from the kinetic observations.

Synthesis of multiacyl poly(ethylene glycol) for the conjugation of cytochrome c to phospholipid vesicle.

To conjugate water-soluble macromolecules on the surface of phospholipid vesicles, we synthesized a poly(ethylene glycol) (PEG)-lipid having four acyl chains using a lysine (Lys)-type monodendron structure. One end of the diamino-PEG was amidified with Lys, and then two amino groups of the Lys moiety were amidified with two Lys derivatives which had been acylated with two stearoyl groups. The other end of the PEG was activated with a triazine group or a pyridyldithio group. The hydrate of the lipid mixture of dipalmitoylphosphatidylcholine, cholesterol, dipalmitoylphosphatidylglycerol, and the PEG-lipid at a molar ratio of 5/5/1/0.3 was extruded in order to prepare the phospholipid vesicles with the average diameter of 270 +/- 20 nm. The coupling ratio of cytochrome c with the PEG-lipid was monitored by HPLC, detecting the pyridyl 2-thione liberated from the pyridyldithio group and determining it to be 26% on the basis of the incorporated PEG-lipid.

Photoreduction of methemoglobin by irradiation in the near-ultraviolet region.

Ferric metHb can be photoreduced to the ferrous state by direct photoexcitation in the near-ultraviolet region. In this research, we studied the mechanism and facilitating conditions for the photoreduction and the resulting restoration of O(2) binding. MetHb in phosphate-buffered saline or pure water in a CO atmosphere was photoreduced to form HbCO by illuminating the N band (365 nm), one of the porphyrin pi --> pi transitions, whereas the photoreduction did not occur in Ar, N(2), or O(2). The transient absorption spectrum exhibited the generation of deoxyHb within 30 ns in both the CO and Ar atmospheres; however, only in CO did the subsequent CO binding inhibit the back reaction. The photoreduction rate was dependent on the pH and ligand anions, showing that aquametHb in the high-spin state was predominant for the photoreduction. Axial ligand-to-metal charge-transfer (LMCT) bands overlap with the Soret and Q bands in metHb; however, the excitation of these bands showed little photoreduction, indicating that the contribution of these LMCT bands is minimal. Excitation of the N band significantly contributes to the photoreduction, and this is facilitated by the external addition of mannitol, hyaluronic acid, Trp, Tyr, etc. Especially, Trp allowed the photoreduction even in an Ar atmosphere, and the reduced Hb can be converted to HbO(2) by O(2) bubbling. One mechanism of the metHb photoreduction that is proposed on the basis of these results consists of a charge transfer from the porphyrin ring to the central ferric iron to form the porphyrin pi cation radical and ferrous iron by the N band excitation, and the contribution of the amino acid residues in the globin chain as an electron donor or an electron pathway.

Molecular dimensions of Hb-based O(2) carriers determine constriction of resistance arteries and hypertension.

The effect of molecular dimension of hemoglobin (Hb)-based O(2) carriers on the diameter of resistance arteries (A(0), 158 +/- 21 microm) and arterial blood pressure were studied in the conscious hamster dorsal skinfold model. Cross-linked Hb (XLHb), polyethylene glycol (PEG)-conjugated Hb, hydroxyethylstarch-conjugated XLHb, polymerized XLHb, and PEG-modified Hb vesicles (PEG-HbV) were synthesized. Their molecular diameters were 7, 22, 47, 68, and 224 nm, respectively. The bolus infusion of 7 ml/kg of XLHb (5 g/dl) caused an immediate hypertension (+34 +/- 13 mmHg at 3 h) with a simultaneous decrease in A(0) diameter (79 +/- 8% of basal value) and a blood flow decrease throughout the microvascular network. The diameter of smaller arterioles did not change significantly. Infusion of larger O(2) carriers resulted in lesser vasoconstriction and hypertension, with PEG-HbV showing the smallest changes. Constriction of resistance arteries was found to be correlated with the level of hypertension, and the responses were proportional to the molecular dimensions of the O(2) carriers. The underlying mechanism is not evident from these experiments; however, it is likely that the effects are related to the diffusion properties of the different Hb molecules.

Constriction of resistance arteries determines l-NAME-induced hypertension in a conscious hamster model.

The influence of infusion of a nitric oxide (NO) synthase inhibitor, N(omega)-nitro-l-arginine methyl ester (l-NAME), on resistance arteries (diameter, 150 +/- 8 microm) and its relationship with hypertension were examined in conscious hamsters fitted with a dorsal skinfold window. After infusing l-NAME (10 and 30 mg/kg), hamsters showed immediate hypertension of +13 +/- 9 and +21 +/- 9 mm Hg, respectively, relative to basal values, and a maximum of +44 +/- 4 mm Hg at 30 min for the high-dose group. There was simultaneous significant vasoconstriction of the resistance arteries (A(0)) which reduced to 60 +/- 5% of baseline diameter at 3 h; however, there was no significant vasoconstriction in large and small arterioles with diameters diameters less than 70 microm. Blood flow rate in all the vessels decreased in consonance with the vasoconstriction of the resistance artery, irrespective of microvessel classification. These results indicate that the resistance artery plays a key role as a regulator and microvascular resistance in determining blood flow distribution and hypertension when a NO synthase inhibitor is infused.