A survey of the current status of neonatal disseminated intravascular coagulation in neonatal intensive care units in Kyushu, Japan.

BACKGROUND: Neonatal disseminated intravascular coagulation (DIC) is a rare disease with a poor outcome. However, data on the incidence, treatment, and outcome of neonatal DIC are scarce. Thus, this study investigated the status of neonatal DIC in Japan. METHODS: We sent a retrospective questionnaire-based survey regarding the status of diagnosis and treatment of neonatal DIC from January 1, 2016, to December 31, 2018, to 30 hospitals in Kyushu with a neonatal-perinatal medicine division. The data collected by the questionnaire survey included information about the patients diagnosed with neonatal DIC. RESULTS: Among the 13,582 neonates surveyed, 120 (0.9 %) were diagnosed with DIC. Of them, clinical data were available for 105 cases. There were 11 deaths (mortality rate: 10.4 %), with the most common underlying condition being infection (n = 9), followed by neonatal asphyxia and hematologic disease (both, n = 1). Compared with the survival group, the death group had more infections, as well as a higher rate of bleeding symptoms and organ dysfunction. CONCLUSIONS: Neonatal DIC associated with infectious diseases has a poor outcome. Therefore, it is necessary to formulate diagnostic and treatment guidelines for early intervention in such cases.

Plasma-derived factor VIIa and factor X mixture agent (MC710) prophylaxis in haemophilia B patients with inhibitors.

INTRODUCTION: Haemophilia B patients with factor IX inhibitors have particularly unmet needs for conventional therapy. AIM: Phase II/III clinical trial, multicentre, open-label, prospective, self-controlled study was conducted to assess MC710 prophylaxis in haemophilia B patients with inhibitors. METHODS: We enrolled haemophilia patients who had received episodic or prophylactic treatment with bypassing agents up to that time. The participants continued their conventional therapy for 24 weeks and then MC710 was prophylactically infused intravenously every 2 or 3 days at 60 to 120 µg as FVIIa per kilogram of body weight for 24 weeks. The primary endpoint was the annual bleeding rate (ABR) requiring bypassing agents, which was compared intraindividually between the conventional therapy period and the MC710 prophylaxis period. RESULTS: A total of 11 male haemophilia B patients were enrolled. The median ABR ratio for each participant (the prophylaxis period ABR divided by the conventional therapy period ABR) was .33 (2.1/6.5), range from .00 to 3.77. ABR ratios for 9 of the 11 patients ranged from .00 to .60, and 3 of the 9 patients had zero bleeding events during the prophylaxis period. Meanwhile, ABR ratios for the remaining two patients were 2.53 and 3.77, respectively. Although a fibrinogen decrease recovered by the dose reduction was reported for only one participant as the sole adverse drug reaction in this study, no thrombotic events or other safety concerns were reported. CONCLUSION: MC710 prophylaxis is considered to be decrease the bleeding rate in haemophilia B patients with inhibitors without safety concerns.

Clinical conditions and risk factors for inhibitor-development in patients with haemophilia: A decade-long prospective cohort study in Japan, J-HIS2 (Japan Hemophilia Inhibitor Study 2).

BACKGROUND: Inhibitor-development is a serious complication in patients with haemophilia (PwH). Previous studies reported that therapeutic and genetic factors could be associated with these alloantibodies. Relevant clinical features such as genetic-background and different treatment regimens in Japan remain unclear, however. AIMS: To analyse a nation-wide Japanese registry for PwH, and to examine risk factors for inhibitor-development. METHODS AND RESULTS: Newly diagnosed patients with haemophilia A (PwHA) or haemophilia B (PwHB) without inhibitors after 2007, and with treatment records traceable from 0 to 75 exposure days (ED), were enrolled in the Japan Hemophilia Inhibitor Study 2 (J-HIS2) initiated in 2008. Of 417 patients (340 PwHA, 77 PwHB) from 46 facilities, 83 (76 PwHA, 7 PwHB) were recorded with inhibitors by July 2020. Inhibitors were observed in 31.0% of severe PwHA, 8.0% moderate and 1.6% mild and in 17.1% of severe PwHB. The majority of inhibitors (89.7% in severe PwHA and 71.4% in severe PwHB) were detected on or before 25ED (median 12ED in PwHA and 19ED in PwHB). Genotyping in these severe patients identified an association between inhibitor-development and null variants of F8 (P < .01) or F9 (P < .05). A lower incidence of inhibitors was recorded in severe PwHA treated with prophylaxis than in those treated on-demand (P < .01). A past-history of intracranial-haemorrhage appeared to be associated with inhibitor-development, while FVIII-concentrates infusion and routine vaccination on the same day was not related to inhibitor-development. CONCLUSION: The J-HIS2 study has identified significant clinical variables associated with inhibitor-development in Japanese PwH, consistent with other global studies.

Spectrum of F8 Genotype and Genetic Impact on Inhibitor Development in Patients with Hemophilia A from Multicenter Cohort Studies (J-HIS) in Japan.

Some genetic and treatment-related factors are risk factors for inhibitor development in patients with hemophilia A (PwHA). However, the genotype distribution of the factor VIII gene (F8) and genetic impact on inhibitor development in Japanese PwHA remain unknown. In 2007, the Japan Hemophilia Inhibitor Study 2 (J-HIS2) was organized to establish a nationwide registry system for hemophiliacs and to elucidate risk factors for inhibitor development, designed for prospective investigation following a retrospective study (J-HIS1) which had already finished. Patients, newly diagnosed after January 2007, were enrolled in J-HIS2 and followed up for inhibitor development and clinical environments since 2008 onward. In the present study, F8 genotypes of PwHA were investigated in the patients recruited from the J-HIS2 cohort as well as those with inhibitor from the J-HIS1 cohort. F8 variants identified in 59 PwHA with inhibitor in J-HIS1 were: 20 intron-22 inversions, 5 intron-1 inversions, 9 large deletions, 4 nonsense, 8 missense, 11 small in/del, and 2 splice-site variants. F8 variants identified in 267 (67 with inhibitor) PwHA in J-HIS2 were: 76(28) intron-22 inversions, 3(2) intron-1 inversion, 1(0) duplication, 8(5) large deletions, 21(7) nonsense, 109(7) missense, 40(11) small in/del, and 9(7) splice-site variants. Forty variants were novel. The cumulative inhibitor incidence rate in the severe group with null changes was 42.4% (95% confidence interval [CI]: 33.7-50.8), higher than that with nonnull changes (15.6% [95%CI: 6.8-27.8]), in J-HIS2. Relative risk for inhibitor development of null changes was 2.89. The spectrum of F8 genotype and genetic impact on inhibitor development in Japanese PwHA were consistent with the previous reports.

Vitamin K Deficiency Bleeding in Infancy.

Vitamin K is essential for the synthesis of few coagulation factors. Infants can easily develop vitamin K deficiency owing to poor placental transfer, low vitamin K content in breast milk, and poor intestinal absorption due to immature gut flora and malabsorption. Vitamin K deficiency bleeding (VKDB) in infancy is classified according to the time of presentation: early (within 24 h), classic (within 1 week after birth), and late (between 2 week and 6 months of age). VKDB in infancy, particularly late-onset VKDB, can be life-threatening. Therefore, all infants, including newborn infants, should receive vitamin K prophylaxis. Exclusive breastfeeding and cholestasis are closely associated with this deficiency and result in late-onset VKDB. Intramuscular prophylactic injections reduce the incidence of early-onset, classic, and late-onset VKDB. However, the prophylaxis strategy has recently been inclined toward oral administration because it is easier, safer, and cheaper to administer than intramuscular injection. Several epidemiological studies have shown that vitamin K oral administration is effective in the prevention of VKDB in infancy; however, the success of oral prophylaxis depends on the protocol regimen and parent compliance. Further national surveillance and studies are warranted to reveal the optimal prophylaxis regimen in term and preterm infants.

Perioperative safety and haematostatic efficacy of a new bypassing agent pd-FVIIa/FX (Byclot) in haemophilia patients with high-responding type inhibitors.

: The novel agent pd-FVIIa/FX is a 1 : 10 protein weight mixture of activated factor VII (FVIIa) and factor X (FX) derived from donated blood plasma. A phase III clinical trial of pd-FVIIa/FX revealed high efficacy for bleeding episodes in haemophilia patients with inhibitors. However, up to now, only one case of this new agent being used for surgery had been reported. The objective of this study is to evaluate the perioperative haemostatic efficacy and safety of pd-FVIIa/FX in haemophilia patients with inhibitors. We retrospectively reviewed 25 operation charts from 14 haemophilia patients with high-responding inhibitors using pd-FVIIa/FX during the perioperative period. Efficacy was evaluated by attending physicians and results divided into four groups (excellent, good, fair, and poor). The operation chart was provided by nine Japanese medical institutes with expertise in haemophilia management. Out of the total of 25 surgical procedures, 44% (11/25) were classified as major surgery and the remainders were minor surgeries. In all of the surgeries but one, rFVIIa and/or APCC were administered in combination or sequential method. In all cases except one, the haemostatic efficiency rate was judged as excellent or good by treating physicians for an overall efficacy rate of 96%. No thrombotic adverse effects were reported. This study's results suggest that both combination and sequential therapy of pd-FVIIa/FX and other bypassing agents are well tolerated and effective for the control of perioperative bleeding in haemophilia patients with high-responding inhibitors.

Incidence and In-Hospital Mortality of Neonatal Disseminated Intravascular Coagulation in Japan: An Observational Study of a Nationwide Hospital Claims Database.

This study aimed to estimate the incidence and prognosis of neonatal disseminated intravascular coagulation (DIC) in Japan by analyzing data retrieved from a national administrative database. Clinically, the prognosis of DIC in neonates is poor, but there is little epidemiological data in Japan. This retrospective observational study identified patients diagnosed with neonatal DIC and who were registered in the Japanese diagnosis procedure combination (DPC) database between April 1, 2014 and March 31, 2016. The patients, who were diagnosed with neonatal DIC, included those with ICD-10 code D65 or P60 in primary and secondary diagnosis, with comorbid conditions existing at admission, and with complications occurring after admission. Of 78,073 neonates admitted to 1,474 neonatal intensive care units, 1,864 (2.4%) were diagnosed with DIC. There was no difference between sexes in incidence of DIC; the incidence of DIC was higher in extremely low birth weight infants (9.8%), and significantly higher than that in normal birth weight infants. The overall mean length of hospital stay was longer in neonates with DIC (69.5 days) than in those without DIC (32.6 days, P < 0.001). The number of deaths was 1,156 (1.5%). In-hospital mortality was significantly higher in neonates with DIC (14.1%) than in those without DIC (1.2%, P < 0.001), especially in premature babies. This nationwide study was the first report to investigate the incidence and in-hospital mortality of neonatal DIC in Japan. Neonatal DIC has a significant impact on prognosis, and its influence is greater in premature than in term infants.

Inhibitor development, safety and efficacy of Advate® among previously treated patients with hemophilia A in a postmarketing surveillance in Japan.

Rurioctocog alfa (recombinant factor VIII: Advate®) is available for the control of bleeding in patients with hemophilia A in Japan. To evaluate the inhibitor development, safety, and efficacy of rurioctocog alfa, a non-interventional and observational postmarketing surveillance was conducted on 352 previously treated Japanese patients aged 1-76 years with ≥ 4 exposure days under the conditions of routine clinical practice. A post-hoc comparison of the mean annualized bleeding rates which required treatment with rurioctocog alfa detected a statistically significant difference (P < 0.0001) between patients treated on regular prophylaxis (8.5 bleeds/year) and patients treated on an on-demand basis (36.6 bleeds/year). Favorable prophylactic and on-demand hemostatic efficacy ("excellent" or "good") were shown in 88.5-100% of patients across all treatment regimens. A total of 22 events of adverse drug reactions were reported in 13 male patients. Of the 352 patients, 3 (0.9%) patients, all of whom had ≤ 50 exposure days before enrollment, developed de novo FVIII inhibitor. No deaths or allergic reactions were reported. Rurioctocog alfa was found to be well-tolerated and effective among patients with hemophilia A in a postmarketing routine clinical practice.

Inhibitor development, safety, and efficacy of Advate® in previously untreated patients with hemophilia A in a postmarketing surveillance in Japan.

Rurioctocog alfa (recombinant Factor VIII: AdvateⓇ) is available for the control of bleeding in patients with hemophilia A in Japan. To evaluate the immunogenicity, safety, and efficacy of prophylactic and on-demand use of rurioctocog alfa, postmarketing surveillance was conducted on 114 previously untreated Japanese patients aged 0-82 years with ≤ 3 exposure days under the conditions of routine clinical practice. A post-hoc comparison of mean annualized bleeding rates between patients in the regular prophylaxis group (7.4 bleeds/year) and in the on-demand treatment group (15.7 bleeds/year) using a negative binomial model found a statistically significant difference (P = 0.0164) in the subset of patients with severe hemophilia A. Favorable prophylactic and on-demand hemostatic efficacy ("excellent" or "good") was shown in 71.4-88.5% across all treatment regimens. A total of 31 events of adverse drug reactions were reported. Of 114 patients, 21 (18.4%) developed de novo FVIII inhibitor; of these, 17 occurred within 50 exposures. One death was reported. A family history of positive inhibitors was significantly associated with inhibitor development (Fisher exact P value = 0.0004); no other risk factors were identified. Rurioctocog alfa was found to be well-tolerated and effective in previously untreated Japanese patients with hemophilia A in this postmarketing surveillance of routine clinical practice.

Correction to: Inhibitor development, safety, and efficacy of Advate® in previously untreated patients with hemophilia A in a postmarketing surveillance in Japan.

The authors would like to correct the errors in the publication of the original article. The correction details are given below.

Perioperative safety and hemostatic efficacy of Advate® in patients with hemophilia A in a postmarketing surveillance in Japan.

Rurioctocog alfa (recombinant factor VIII: Advate®) is available for the control of bleeding among patients with hemophilia A in Japan. To evaluate the perioperative safety and hemostatic efficacy of Advate®, a postmarketing surveillance was conducted in Japanese patients undergoing surgery in a real-world setting. A total of 74 surgical procedures performed in 58 subjects aged 0-75 years, including three females, were studied. A hemostatic efficacy rating of "excellent" or "good" was reported in 73/74 surgical procedures (98.6%). Perioperative bleeding was successfully controlled by Advate® in five subjects with positive FVIII inhibitors (2.4-9.1 BU/mL). Advate® was administered at higher initial bolus doses (114-385 IU/kg) and at higher rates by subsequent initial continuous infusion (8.3-15 IU/kg/hour) in the five subjects with inhibitor than in the subjects without inhibitor (n = 47; mean initial bolus dose: 53.4 IU/kg; subsequent mean initial continuous infusion: 3.8 IU/kg/h). Adverse drug reactions were reported in 7/74 (9.5%) procedures, two of which were the development of de novo FVIII inhibitors. Overall, the perioperative use of Advate® in a real-world setting was found to be safe and effective among Japanese patients with hemophilia A.

N1-Nonyl-1,4-diaminobutane ameliorates brain infarction size in photochemically induced thrombosis model mice.

Inhibitors for polyamine oxidizing enzymes, spermine oxidase (SMOX) and N1-acetylpolyamine oxidase (PAOX), were designed and evaluated for their effectiveness in a photochemically induced thrombosis (PIT) mouse model. N1-Nonyl-1,4-diaminobutane (C9-4) and N1-tridecyl-1,4-diaminobutane (C13-4) competitively inhibited the activity of PAOX and SMOX in a manner comparable to N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL72527), an irreversible inhibitor of both enzymes. The two compounds were then tested for their effects in the PIT model. Both intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) administration of C9-4 decreased infarct volumes significantly. By contrast, C13-4 reduced the volume of brain infarction by i.c.v. administration, but no reduction was observed after i.p. administration. C9-4 administered by i.p. injection reduced the volume of brain infarction significantly at doses of more than 3 mg/kg, and the dosage of 5 mg/kg or 10 mg/kg demonstrated the most potent effect and were more effective than equivalent doses of the other inhibitors such as MDL72527 and N-benzylhydroxylamine. I.P. injection of 5 mg/kg of C9-4 provided a therapeutic time window of longer than 12 h. This report demonstrates that C9-4 is a potent inhibitor of the polyamine oxidizing enzymes and is useful lead compound for candidate drugs with a long therapeutic time window, to be used in the treatment of ischemic stroke.

Synthesis and evaluation of fatty acid amides on the N-oleoylethanolamide-like activation of peroxisome proliferator activated receptor α.

A series of fatty acid amides were synthesized and their peroxisome proliferator-activated receptor α (PPAR-α) agonistic activities were evaluated in a normal rat liver cell line, clone 9. The mRNAs of the PPAR-α downstream genes, carnitine-palmitoyltransferase-1 and mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase, were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR) as PPAR-α agonistic activities. We prepared nine oleic acid amides. Their PPAR-α agonistic activities were, in decreasing order, N-oleoylhistamine (OLHA), N-oleoylglycine, Oleamide, N-oleoyltyramine, N-oleoylsertonin, and Olvanil. The highest activity was found with OLHA. We prepared and evaluated nine N-acylhistamines (N-acyl-HAs). Of these, OLHA, C16:0-HA, and C18:1Δ(9)-trans-HA showed similar activity. Activity due to the different chain length of the saturated fatty acid peaked at C16:0-HA. The PPAR-α antagonist, GW6471, inhibited the induction of the PPAR-α downstream genes by OLHA and N-oleoylethanolamide (OEA). These data suggest that N-acyl-HAs could be considered new PPAR-α agonists.

The International Immune Tolerance Induction Study and its follow-up study on Japanese hemophilia A patients with inhibitors.

The International Immune Tolerance Induction (I-ITI) Study in hemophilia A patients with inhibitors included 16 Japanese patients among a total of 115 test subjects. The results within this group of Japanese patients were 11 cases of I-ITI off-study, three cases of I-ITI on-study, and two cases of tolerance on prophylaxis. There was no significant difference in success rate between the low-dose and high-dose groups (Study I). Successively, independent follow-up survey in Japan was conducted in 14 cases, with consent (Study II). Ten cases were off-study at the end of the I-ITI Study. Of these 10 cases, seven of seven successful cases remained clinical successes at the end of the follow-up study, one partial success became a full success while a second relapsed, and one failure was subsequently evaluated as a partial success. Four cases that were on-study at the end of I-ITI Study were classified as three successes and one failure at the end of the follow-up study. As a result, the status at the end of follow-up study was: 11 ITI successes (78.6 %); one partial success; one failure; and one relapse. Thus, the ITI follow-up study was helpful in providing a long-term prognostic determination of inhibitors.

Long-term follow-up of children with refractory immune thrombocytopenia treated with rituximab.

Data on long-term outcomes of children with refractory immune thrombocytopenia (ITP) treated with rituximab are limited. We retrospectively analyzed the long-term effect of rituximab on 22 pediatric ITP patients (11 boys and 11 girls). Compete response (CR) (platelet count ≥100 × 10(9)/L) and partial response (PR) (platelet count 30-99 × 10(9)/L) were achieved in nine (41 %) and two (9 %) patients, respectively. Of the 11 responders, eight subsequently relapsed 2-26 months after initial rituximab treatment. The 5-year relapse-free rate was 14 % (3/22, 95 % confidence interval: 0-27 %) with a median follow-up period of 6.4 years. Five initial responders with subsequent relapse and one non-responder received multiple rituximab treatments of nine courses; all patients responded to the second rituximab therapy without any significant toxicity. All eight patients who relapsed after an initial response and six of 11 non-responders achieved CR or PR with subsequent treatment, including repeated courses of rituximab, splenectomy, steroids, and other immunomodulating agents. Our findings indicated that the sustained effect of rituximab on children with refractory ITP is low, but that the long-term outcome of ITP itself is not poor. Furthermore, repeated rituximab administration may be a promising therapy for those who relapse after an initial response.

Postmarketing Surveillance of Recombinant Human Soluble Thrombomodulin (Thrombomodulin α) in Pediatric Patients With Disseminated Intravascular Coagulation.

Recombinant human soluble thrombomodulin (thrombomodulin α [TM-α]) has been marketed as a novel anticoagulant for disseminated intravascular coagulation (DIC) in Japan since 2008. Postmarketing surveillance (PMS) has been conducted since its approval. As effectiveness and safety were not previously determined in pediatric patients, this study evaluated PMS data and examined the usefulness of TM-α in treating pediatric DIC. After excluding newborn infants, data for 210 pediatric patients were analyzed and compared to 3786 adult patients. The day after the last TM-α administration, DIC had resolved in 58.5% of the patients. At 28 days after the last TM-α administration, the survival rate was 71.6%. Nineteen episodes of adverse drug reactions were observed in 11 patients but no significant differences were noted for effectiveness and safety. Although this study was limited by its retrospective design, including selection biases and no limitation on concomitant use of other anticoagulants, TM-α appears to be useful for the treatment of DIC in both pediatric and adult patients.

Recombinant soluble human thrombomodulin (thrombomodulin alfa) in the treatment of neonatal disseminated intravascular coagulation.

UNLABELLED: Recombinant soluble human thrombomodulin (TM-α) has been shown to be useful in the treatment of disseminated intravascular coagulation (DIC) in a heparin-controlled study and has been available for clinical use in Japan since 2008. However, data on its use for neonatal DIC have not been reported from any clinical studies, so efficacy and safety were analyzed in 60 neonatal DIC patients identified in post-marketing surveillance. The DIC resolution rate as of the day after last administration of TM-α was 47.1 %, and the survival rate at 28 days after last administration was 76.7 %. Hemostatic test result profiles revealed decreased levels of fibrin/fibrinogen degradation products and increased platelet counts and antithrombin activity. Incidences of adverse drug reactions, bleeding-related adverse drug reactions, and bleeding-related adverse events were 6.7, 6.7, and 16.7 %, respectively, with no significant differences between neonatal, pediatric (excluding neonates), and adult DIC patients. CONCLUSION: This surveillance provided real-world data on the safety and effectiveness of TM-alpha in the treatment of neonatal DIC in general practice settings.

Modulatory effects of Bifidobacterium longum BB536 on defecation in elderly patients receiving enteral feeding.

AIM: To investigate the effects of the probiotic Bifidobacterium longum BB536 on the health management of elderly patients receiving enteral feeding. METHODS: Two double-blind, placebo-controlled trials were performed with long-term inpatients receiving enteral tube feeding at Kitakyushu Hospital Group, Fukuoka, Japan. BB536 was administered as BB536-L and BB536-H powders that contained approximately 2.5 × 10¹° and 5 × 10¹° cfu of BB536, respectively. In the first trial, 83 patients (age range: 67-101 years) were randomized into 2 groups that received placebo (placebo group) or BB536-H (BB536 group) powders. In the second trial, 123 patients (age range: 65-102 years) were randomized into 3 groups, and each group received placebo (placebo group), BB536-L (BB536-L group), or BB536-H (BB536-H group) powders. Each patient received the study medication for 16 wk after 1 wk of pre-observation. Fecal samples were collected from each patient prior to and after the intervention during Trial 2. Clinical observations included body temperature, occurrence of infection, frequency of defecation, and fecal microbiota. RESULTS: No significant changes were observed in the frequency of defecation for either treatment in Trial 1. However, a significant change was noted in the BB536-L group (P = 0.0439) in Trial 2 but not in the placebo or BB536-H groups. Subgroup analyses based on the frequency of defecation for each patient during the pre-observation period for both trials revealed significant increases in bowel movements in patients with a low frequency of defecation and significant decreases in the bowel movements of patients with a high frequency of defecation during the intervention period in the BB536 groups. The combination of Trials 1 and 2 data revealed a modulatory effect of BB536 ingestion on the changes in bowel movements. Significantly increased bowel movements were observed in patients in the low frequency subgroup with significant intergroup differences (P < 0.01). Significantly decreased bowel movements were observed in patients in the high subgroup, but no significant intergroup differences were observed compared with the placebo group. BB536 ingestion increased the prevalence of normally formed stools. BB536 intake also significantly (P < 0.01) increased the cell numbers of bifidobacteria in fecal microbiota, and significant intergroup differences were observed at week 16. No adverse events were reported in any group. CONCLUSION: Our results suggest that BB536 ingestion modulated the intestinal environment and may have improved the health care of elderly patients receiving enteral feeding.

Pentamines as substrate for human spermine oxidase.

Substrate activities of various linear polyamines to human spermine oxidase (hSMO) were investigated. The activities were evaluated by monitoring the amount of H2O2 released from sample polyamines by hSMO. H2O2 was measured by a HPLC method that analyzed fluorescent dimers derived from the oxidation of homovanillic acid in the presence of horseradish peroxidase. Six triamines were tested and were found not to be hSMO substrates. Of sixteen tetramines tested, spermine (Spm) was the most active substrate, followed by homospermine and N-butylated Spm. Pentamines showed a characteristic pattern of substrate activity. Of thirteen pentamines tested, 3343 showed higher substrate activity than Spm, and 4343 showed similar activity to Spm. The activities of the other pentamines were as follows: 3443, 4443, 4344, 3344, 4334, 4444, and 3334 (in decreasing order). Product amines released from these pentamines by hSMO were then analyzed by HPLC. Triamine was the only observed product, and the amount of triamine was nearly equivalent to that of released H2O2. A marked difference in the pH dependency curves between tetramines and pentamines suggested that hSMO favored reactions with a non-protonated secondary nitrogen at the cleavage site. The Km and Vmax values for Spm and 3343 at pH 7.0 and 9.0 were consistent with the higher substrate activity of 3343 compared to Spm, as well as with the concept of a non-protonated secondary nitrogen at the cleavage site being preferred, and 3343 was well degraded at a physiological pH by hSMO.