Yutaka Niihara

Aged Garlic Extract May Be Safe for Patients on Warfarin Therapy

Garlic has been known to have antiplatelet properties. Because of the lack of major clinical data regarding the safety of concomitant use of garlic supplements and anticoagulants, we decided to evaluate the safety of using garlic extract along with oral anticoagulation therapy. During this project we tested aged garlic extract (AGE), a commercial garlic preparation, with warfarin (Coumadin). Sixty-six (66) patients were screened for a double-blind, randomized, placebo-controlled pilot study. Fifty-two (52) patients were randomized for the project. Forty-eight patients (30 men and 18 women, with a mean age of 56+/-10 years) completed the study. Eighteen patients (14 before randomization, 4 after randomization) were dropped from the study. The study medication (AGE or placebo) was administered at a dose of 5 mL twice a day for 12 wk. Potential bleeding and thromboembolic episodes were monitored. There was no evidence of increased hemorrhage in either the placebo or the AGE group. Adverse events included headache, fatigue, colds, and dizziness. However, no significant difference was found in the incidence of these minor adverse events between the groups. Thus, the adverse events are unlikely to be attributable to AGE. The results suggest that AGE is relatively safe and poses no serious hemorrhagic risk for closely monitored patients on warfarin oral anticoagulation therapy. Although the risk-benefit ratio of AGE use needs to be considered carefully when warfarin therapy is necessary, its positive effects may be beneficial to people with a high-risk background or who are taking cardiovascular medications.

Oral L-glutamine therapy for sickle cell anemia: I. Subjective clinical improvement and favorable change in red cell NAD redox potential.

Previously, we demonstrated that there is an increased utilization of glutamine by intact sickle red blood cells (RBC) in conjunction with nicotinamide adenine dinucleotide (NAD) metabolism in vitro. In this report, we describe the in vivo effect of L‐glutamine supplementation on total NAD, nicotinamide adenine dinucleotide reduced (NADH), and NAD redox potential of sickle RBC. Seven adult sickle cell anemia patients participated in this study. The exclusion criteria were pregnancy, previous or current use of hydroxyurea, and transfusion within 3 months of initiation of the study. After proper consent, L‐glutamine was started at a dose of 30 g/day administered orally. Fasting blood samples were drawn at baseline and after 4 weeks of therapy by routine phlebotomy for evaluation of RBC total NAD and NADH levels. We found significant changes in both the NADH level and NAD redox potential (ratio of NADH to NAD+ + NADH). NAD redox potential increased from 47.2 ± 3.7% to 62.1 ± 11.8% (P < 0.01). The NADH level increased from 47.5 ± 6.3 to 72.1 ± 15.1 nmol/ml RBC (P < 0.01). The total NAD level demonstrated an upward trend (from 101.2 ± 16 to 116.4 ± 14.7 nmol/ml RBC) but this was not statistically significant. Our data show that oral L‐glutamine can significantly increase the NAD redox potential and NADH level in sickle RBC. These changes may decrease oxidative susceptibility of sickle RBC and result in clinical benefit. Am. J. Hematol. 58:117–121, 1998.

Increased red cell glutamine availability in sickle cell anemia: Demonstration of increased active transport, affinity, and increased glutamate level in intact red cells

Sickle red blood cells (RBCs) have been shown to have an increase in total nicotinamide adenine dinucleotide (NAD) content by an as-yet-unknown mechanism. Because glutamine is an essential precursor in NAD biosynthesis, we have examined the rates of active RBC glutamine transport and glutamine transport kinetics with Michaelis-Menten constant (K[m]) and maximum velocity (V[max]) in RBCs from patients with sickle cell disease, patients with high reticulocyte counts, and normal volunteers. In addition, plasma and RBC levels of glutamine and glutamate in the three groups were analyzed. The rate of active glutamate transport in sickle RBCs increased threefold over that in high-reticulocyte RBCs and increased 15-fold over that in normal RBCs. Glutamine transport K(m) in sickle RBCs was decreased fivefold in comparison with that in the high-reticulocyte group and that in normal control subjects. Glutamine transport V(max) for sickle RBCs was twofold and eightfold higher in comparison with those in the high-reticulocyte RBCs and normal control RBCs, respectively. Finally, the level of RBC glutamate (a byproduct of glutamine in NAD synthesis) in the sickle group was significantly increased in comparison with that in the high-reticulocyte group, whereas the RBC glutamine level was not. The higher glutamate level in sickle cells may suggest a higher glutamine turnover in these cells. These data suggest that sickle RBCs have an increased glutamine availability and affinity that may facilitate the increase in total NAD in sickle RBCs.

Aged garlic extract therapy for sickle cell anemia patients

BackgroundSickle cell anemia is one of the most prevalent hereditary disorders with prominent morbidity and mortality. With this disorder oxidative, phenomena play a significant role in its pathophysiology. One of the garlic (Allium sativum L.) formulations, aged garlic extract (AGE), has been reported to exert an anti-oxidant effect in vitro, we have evaluated the anti-oxidant effect of AGE on sickle red blood cells (RBC).MethodsFive patients (two men and three women, mean age 40 ± 15 years, range 24–58 years) with sickle cell anemia participated in the study. AGE was administered at a dose of 5 ml a day. Whole blood samples were obtained at baseline and at 4 weeks for primarily Heinz body analysis.ResultsThe data were consistent with our hypothesis. In all patients, the number of Heinz bodies decreased over the 4 week period (58.9 ± 20.0% at baseline to 29.8 ± 15.3% at follow-up, p = 0.03).ConclusionsThese data suggest that there is a significant anti-oxidant activity of AGE on sickle RBC. AGE may be further evaluated as a potential therapeutic agent to ameliorate complications of sickle cell anemia.

A Phase 3 Study of L-Glutamine Therapy for Sickle Cell Anemia and Sickle ß0-Thalassemia

H Risk Papillomavirus (hr-HPV) genotypes are causal agents for cervical cancer and some geographical variation has been reported. Available vaccines cater for up to nine hr-HPV genotypes, which may not necessarily be the most predominant strains in every region of the world. Data on HPV genotype in South African regions is limited and unavailable for KwaZuluNatal (KZN) province .Our study aimed to describe hr-HPV genotype distribution among young women in relation to cervical cancer vaccination. A total of 1223 sexually active young women were recruited from high schools, 658 (54%) were from Ugu and 565 (46%) from Ilembe districts and these were investigated for high risk Human Papillomavirus, using GP5/6+ primers and enzyme immunoassay. Of the 1223 vaginal lavages, 301 (25%) were positive for hr-HPV. Of these, 177 (27%) and 124 (22%) were from Ugu and Ilembe districts respectively (p = 0.046). HPV type distribution per district revealed an overall similar distribution, the most predominant types in descending order were HPV 16, 51, 18, 35, 33, 56, 45, 52 and 59. Our data describes high-risk HPV strains infecting young women in the KwaZulu-Natal province and demonstrated that the new nanovalent HPV vaccine does not protect against all predominant hrHPV types. HPV 51, 35 and 56 are most prevalent in KZN yet not covered by the nanovalent vaccine. Investigation of cervical cancer cases would be necessary to ascertain that these types are involved in a significant proportion of these cases.Background: The tumor suppressor phosphatase and tensin homolog (PTEN) is a pleiotropic enzyme, inhibiting phosphatidyl-inositol-3 kinase (PI3K) signaling in the cytosol and stabilizing the genome in the nucleus. Nucleo-cytosolic partitioning is dependent on the posttranslational modifications ubiquitinylation and sumoylation. This cellular compartmentalization of PTEN was investigated in lung neuro-endocrine tumors (lung NET). Methods: Tumor tissues from 192 lung NET patients (surgical specimens=183, autopsies=9) were investigated on tissue microarrays. PTEN was H-scored by two investigators in nucleus and cytosol using the monoclonal antibody 6H2.1. Results were correlated with immunoreactivity for USP7 (herpes virus-associated ubiquitin-specific protease 7) and SUMO2/3 (small ubiquitin-related modifier protein 2/3) as well as PTEN and p53FISH gene status. Clinico-pathologic data including overall survival, proliferation rate and diagnostic markers (synaptophysin, chromogranin A, Mib-1, TTF-1) were recorded. Results: The multicentre cohort included 58 typical carcinoids (TC), 42 atypical carcinoids (AC), 32 large cell neuro-endocrine carcinomas (LCNEC) and 60 small cell lung carcinomas (SCLC). Carcinoids were smaller in size and had higher synaptophysin and chromogranin-A, but lower TTF-1 expressions. Patients with carcinoids were predominantly female and 10 years younger than patients with LCNEC/SCLC. In comparison to the carcinoids, LCNEC/SCLC tumors presented a stronger loss of nuclear and cytosolic PTEN associated with a loss of PTEN and p53. Concomitantly, a loss of nuclear USP7 but increase of nuclear and cytosolic SUMO2/3 was found. Loss of nuclear and cytosolic PTEN, loss of nuclear USP7 and increase of cytosolic SUMO2/3 thus correlated with poor survival. Among carcinoids, loss of cytosolic PTEN was predominantly found in TTF1-negative larger tumors of male patients. Among SCLC, loss of both cytosolic and nuclear PTEN but not proliferation rate or tumor size delineated a subgroup with poorer survival (all p-values <0.05). Conclusions: Cellular ubiquitinylation and sumoylation likely influence the functional PTEN loss in high grade lung NET. Both nuclear and cytosolic PTEN immunoreactivity should be considered for correlation with clinico-pathologic parameters.Background: New treatments for patients with sickle cell disease (SCD) are needed. Oxidative stress may lead to disturbance of cell membranes, exposure of adhesion molecules and damage to the contents of the sickle red blood cells (s-RBC). Nicotinamide adenine dinucleotide (NAD) molecules modulate oxidation-reduction in s-RBCs. Our previous laboratory work demonstrated enhancement of NAD in s-RBC by supplementing a precursor of NAD, L-glutamine. In our Phase-2 clinical study in SCD, oral prescription grade L-glutamine (PGLG) signaled a decreasing trend for painful crises at 24 weeks and a significant decrease in hospitalization at 24 weeks. Methods: A randomized (2:1) Phase 3 placebo-controlled trial was conducted across the United States. Subjects were stratified by hydroxyurea usage. Eligibility criteria included patients ≥5 years of age with diagnoses of HbSS or HbS/β 0 -thalassemia with at least two episodes of sickle cell crises (SCC) during the 12 months prior to screening. PGLG at 0.6 g/kg/day (max 30 g), or placebo, was self-administered in two divided doses orally. The primary endpoint was number of SCC; secondary endpoints included rates of hospitalization and adverse events; additional analyses included cumulative hospital days, incidence of acute chest syndrome (ACS) and time to first crises. Results: A total of 230 patients were enrolled at 31 sites. Groups were well balanced for clinical characteristics. The median incidence of SCC (3 vs. 4 events; p=0.008) as well as hospitalizations (2 events vs. 3; p=0.005) was significantly lower in the treatment group compared to the placebo group. Median cumulative hospital days were lower by 41% in the treatment group (6.5 days) vs. the placebo group (11 days) (p=0.022); ACS was 11.9% in the treatment group and 26.9% in the placebo group (p=0.006). The median time to first crisis was 87 days in the treatment group vs. 54 days in placebo group (p=0.010). Analysis by hydroxurea use, age, and gender yielded consistent findings. Adverse events in the treatment arms were similar between groups. Conclusion: This Phase-3 study in SCD demonstrated that treatment with PGLG provided clinical benefit over placebo by reducing the frequency of painful crises and hospitalization. Additional benefit was observed when evaluating ACS, time to first crises and duration of hospital stay. PGLG was relatively easy to administer and did not require special monitoring.The oncofetal H19 long non-coding RNA (lnc RNA) is normally expressed in the embryo and down regulated at birth; however its expression is re-elicited in human tumors of almost every type. Accumulating data by us and others consolidate into a paradigm according to which H19 is in the center of a mammalian “selfish survival pathway” activated by the cancerous cell in order to cope with stress conditions. P53 nullification or dysfunction, hypoxia, serum starvation, chemotherapy and other stress inducers up-regulate H19 expression. H19 in turn supports tumor growth and enhances proliferation in response to hypoxia and p53 mutations. We have also recently proven that H19 RNA significantly contributes to epithelial to mesenchymal transition (EMT), a process which is known to emerge as a response to a multitude of stress conditions. Surprisingly, studies indicate that H19 is involved also in the further, apparently converse steps of mesenchymal to epithelial transition to support colonization and proliferation in the secondary tumor site. In view of the paradigm suggested above, H19’s unique expression in cancers and the active involvement of H19 in almost every deleterious aspect of cancer progression, H19 should serve as a state of the art target for highly selective cancer therapy. Indeed, we currently use a DNA-plasmid based drug that harnesses H19 regulatory elements to drive the expression of Diphtheria toxin – a translational regulated therapy designed to selectively kill cancer cells with no collateral damage to the healthy surrounding tissues. We will comprehensively review recent studies that align with our paradigm and demonstrate how understanding of H19 expression and biology is translated into promising results in bladder, pancreas and ovarian cancer clinical trials.

Aged Garlic Extract Is a Potential Therapy for Sickle-Cell Anemia

Sickle-cell anemia is one of the most prevalent hereditary disorders with prominent morbidity and mortality. Oxidative phenomena play a significant role in the disorders pathophysiology. A forumlation of garlic (Allium sativum), AGE, has been reported to exert an antioxidant effect in vitro. We evaluated the antioxidant effect of AGE on sickle red blood cells (RBCs). Five patients (two men and three women, mean age 40+/-15 years, range 24-58 years) with sickle-cell anemia participated in the study. AGE was administered at a dose of 5 mL daily. Whole blood samples were obtained at baseline and at 4 wk, primarily for Heinz body analysis. In all patients, the number of Heinz bodies decreased over the 4-wk period (58.9+/-20.0% at baseline to 29.8+/-15.3% at follow-up; P=0.03). These data suggest that AGE has a significant antioxidant activity on sickle RBCs. AGE may be further evaluated as a potential therapeutic agent to ameliorate complications of sickle-cell anemia.

Desferrioxamine decreases NAD redox potential of intact red blood cells: evidence for desferrioxamine as an inducer of oxidant stress in red blood cells

BackgroundDesferrioxamine (DFO) is an important iron chelating agent. It has also been thought of as an agent with anti-oxidant potential as it chelates ferric iron in various parts of the body. However, there is evidence suggesting that it may paradoxically affect red blood cells (RBC) by inducing intracellular oxidant stress. To further understand the mechanism of DFOs interaction with RBC, we conducted a study to determine the effect of DFO upon RBCs redox status.MethodsWe examined NAD redox potential in intact RBC (N = 5) incubated with DFO. RBC were incubated with 6 mM DFO for 2 hours.ResultsSignificant decreases in NAD redox potential were observed after incubation of RBC with 6 mM DFO. The mean decrease was 10.01 ± 1.98% (p < 0.0004).ConclusionsThe data confirm the oxidant effect of DFO on RBC.

L-Glutamine Therapy Reduces Hospitalization for Sickle Cell Anemia andSickle òð-Thalassemia Patients at Six Months â A Phase II RandomizedTrial

Background: Increased oxidant stress plays an important role in the pathophysiology of sickle cell disease. Nicotinamide Adenine Dinucleotide (NAD) is an important anti-oxidant that protects hemoglobin as demonstrated in diseases such as methemoglobinemia. Early in-vitro studies have shown that L-glutamine, a precursor for NAD, reduced oxidant stress via improvement of NAD redox status in red blood cells.Oral administration of L-glutamine in early clinical studies supported in-vitro findings of improving NAD redox potential, therefore, a larger proof of concept clinical trial was designed and conducted. nMethods: A Phase II randomized, double-blind, placebo-controlled, parallel-group, multicenter study was conducted to evaluate the safety and efficacy of L-glutamine therapy for patients 5 years or older diagnosed with sickle cell anemia or sickle β°-thalassemia. Eighty one patients were randomized (1:1 ratio) to oral L-glutamine at 0.3 g/kg or placebo twice daily for 48 weeks. The primary endpoint was the frequency of painful crises. Secondary endpoints included the frequency of hospitalization. nResults: At Week 24 (6 months), the mean number of painful crises was 2.5 and 5.5 for L-glutamine and placebo groups respectively (p = 0.060). The mean number of hospitalizations was 0.8 and 1.3 for L-glutamine and placebo groups respectively (p = 0.036). nConclusion: At 6 months of therapy, L-glutamine treatment was efficacious in reducing the frequency of hospitalization (nearly 40% reduction) and there was a major trend for the decrease in frequency of painful crises (over 50% reduction) favoring the L-glutamine treatment arm. There was no difference in safety between groups. Based on these findings, a Phase III trial was conducted and results are now available.

Desferrioxamine (DFO) conjugated with starch decreases NAD redox potential of intact red blood cells (RBC): Evidence for DFO as an extracellular inducer of oxidant stress in RBC

Desferrioxamine (DFO) is an important iron‐chelating agent. It has also been thought of as an agent with anti‐oxidant potential as it chelates ferric iron in various parts of the body. However, there is evidence suggesting that it may paradoxically affect red blood cells (RBCs) by inducing intracellular oxidant stress. Recently we observed that incubation of RBCs with DFO decreases NAD redox potential in normal RBC. To further understand the mechanism of DFOs interaction with RBC, we conducted a study to determine the effect of extracellular DFO upon RBCs redox status. We examined NAD redox potential in intact RBC (N = 7) incubated with DFO conjugated to starch. RBCs were incubated with 4 mM DFO for 3½ hr and with 6 mM DFO for 2 and 3½ hr. Significant decreases in NAD redox potential were observed after the incubations. With 4 mM DFO at the 3 ½ hr time point the mean decrease was 12.37% ± 9.96% (P < 0.0085). With 6 mM DFO, the mean decreases were 18.54% ± 9.79% (P < 0.0013) and 19.16% ± 8.78% (P < 0.0006) for the 2 and 3 ½ hr incubations, respectively. DFO by itself is very poorly permeable to RBC. Conjugation with starch further ensured impermeability of DFO. The data presented here confirm the oxidant effect of DFO on RBC. The data also demonstrate that the effect of DFO on RBCs NAD redox potential originates extracellularly. Am. J. Hematol. 65:281–284, 2000.

A phase 3 trial of l-glutamine in sickle cell disease

BACKGROUND Oxidative stress contributes to the complex pathophysiology of sickle cell disease. Oral therapy with pharmaceutical‐grade l‐glutamine (USAN, glutamine) has been shown to increase the proportion of the reduced form of nicotinamide adenine dinucleotides in sickle cell erythrocytes, which probably reduces oxidative stress and could result in fewer episodes of sickle cell–related pain. METHODS In a multicenter, randomized, placebo‐controlled, double‐blind, phase 3 trial, we tested the efficacy of pharmaceutical‐grade l‐glutamine (0.3 g per kilogram of body weight per dose) administered twice daily by mouth, as compared with placebo, in reducing the incidence of pain crises among patients with sickle cell anemia or sickle β0‐thalassemia and a history of two or more pain crises during the previous year. Patients who were receiving hydroxyurea at a dose that had been stable for at least 3 months before screening continued that therapy through the 48‐week treatment period. RESULTS A total of 230 patients (age range, 5 to 58 years; 53.9% female) were randomly assigned, in a 2:1 ratio, to receive l‐glutamine (152 patients) or placebo (78 patients). The patients in the l‐glutamine group had significantly fewer pain crises than those in the placebo group (P=0.005), with a median of 3.0 in the l‐glutamine group and 4.0 in the placebo group. Fewer hospitalizations occurred in the l‐glutamine group than in the placebo group (P=0.005), with a median of 2.0 in the l‐glutamine group and 3.0 in the placebo group. Two thirds of the patients in both trial groups received concomitant hydroxyurea. Low‐grade nausea, noncardiac chest pain, fatigue, and musculoskeletal pain occurred more frequently in the l‐glutamine group than in the placebo group. CONCLUSIONS Among children and adults with sickle cell anemia, the median number of pain crises over 48 weeks was lower among those who received oral therapy with l‐glutamine, administered alone or with hydroxyurea, than among those who received placebo, with or without hydroxyurea. (Funded by Emmaus Medical; ClinicalTrials.gov number, NCT01179217.)