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R W Pero

Publications and source records attributed to R W Pero.

At least 19 recordsLinked to original sources

C-Med 100, a hot water extract of Uncaria tomentosa, prolongs lymphocyte survival in vivo.

Water extracts of the bark of Uncaria tomentosa, a vine indigenous to South America, has been used for generations as an "immuno modulator". To understand the basis of this immuno modulatory effect we fed mice in their drinking water with C-Med 100, which is a commercially available water extract from Uncaria tomentosa. We found a dose-dependent increase in spleen cell numbers in the supplemented mice, but the proportions of B cells, T cells, NK cells, granulocytes, and memory lymphocytes were normal. However, there were no detectable changes of the lymphoid architecture of the spleen even after long-term treatment. Further, when C-Med 100 treatment was interrupted the cellularity returned to normal level within four weeks. The increased number of lymphocytes was most likely not due to increased production because C-Med 100 did not have any significant effect on precursor cells nor on the accumulation of recent thymic emigrants in the spleen. We conclude that accumulation is most likely due to prolonged cell survival, because adoptive transfer experiments demonstrated that C-Med 100 treatment significantly prolonged lymphocyte survival in peripheral lymphoid organs, without increasing their proliferation rate. Since the accumulation was reversible and without detectable pathological effects, these results suggest the use of C-Med 100 as a potential agent for clinically accelerating the recovery of patients from leukopenia.

Adjuvants, Immunologic↗

Mechanism of action for N-substituted benzamide-induced apoptosis.

We have analysed the mechanism of action for induction of apoptosis by N-substituted benzamides using declopramide as a lead compound. We show here that declopramide at doses above 250 microM in the mouse 70Z/3 pre-B cell line or in the human promyeolocytic cancer cell line HL60 induced cytochrome c release into the cytosol and caspase-9 activation. The broad spectrum caspase inhibitor zVADfmk and caspase-9 inhibitor zLEDHfmk inhibited apoptosis and improved cell viability when administrated to cells 1 h before exposure to declopramide, whereas the caspase-8 inhibitor zIEDHfmk had less effect. Also, the over expression of Bcl-2 by transfection in 70Z/3 cells inhibited declopramide-induced apoptosis. Prior to the induction of apoptosis, a G(2)/M cell cycle block was induced by declopramide. The cell cycle block was also observed in the presence of broad spectrum caspase inhibitor zVADfmk and in a transfectant expressing high levels of Bcl-2. Furthermore, while p53 was induced in 70Z/3 cells by declopramide, neither the apoptotic mechanism nor the G(2)/M cell cycle block were dependent on p53 activation since both effects were also seen in p53 deficient HL60 cells after addition of declopramide.

Apoptosis↗

N-substituted benzamides inhibit nuclear factor-kappaB and nuclear factor of activated T cells activity while inducing activator protein 1 activity in T lymphocytes.

N-substituted benzamides are compounds that have recently been reported to inhibit nuclear factor-kappaB (NF-kappaB) activity and induce apoptosis in a pre-B cell line. In this study, we focused on the effects of N-substituted benzamides on transcriptional regulation in Jurkat T cells. We used a model system where the cells can be stimulated either through TCR/CD28 or by treatment of the cells with PMA and ionomycin to induce transcription factors typical for T lymphocyte activation. Treatment of the Jurkat cells with procainamide did not influence the transcription factor profile of stimulated cells, while treatment with a derivative having an acetyl group in position 4 of the aromatic ring inhibited NF-kappaB and nuclear factor of activated T cells (NFAT) activity. Declopramide, which contains a chloride in position 3 of the aromatic ring, was inactive in this system, whereas also the acetylated derivative of this compound inhibited NF-kappaB and NFAT activity. In contrast, the transcriptional activity and nuclear expression of activator protein 1 induced by TCR/CD28 stimulation or PMA and ionomycin treatment was enhanced by the acetylated variants of the N-substituted benzamides. Finally, we investigated the effect of N-substituted benzamides on intact promoters for two genes central in immune regulation; the CD40 ligand (CD40L) and IL-2 promoters. The transcriptional activity of the CD40L promoter as well as surface expression of the CD40L induced by signaling through TCR/CD28 was inhibited by addition of acetylated N-substituted benzamides, while the transcriptional activity of the IL-2 promoter was enhanced. Taken together, these data indicate that derivatives of N-substituted benzamides are potential drug candidates for quantitative as well as qualitative modulation of immune functions.

Acecainide↗

DNA repair enhancement of aqueous extracts of Uncaria tomentosa in a human volunteer study.

The Uncaria tomentosa water extracts (C-Med-100) have been shown to enhance DNA repair, mitogenic response and leukocyte recovery after chemotherapy-induced DNA damage in vivo. In this study, the effect of C-Med-100 supplement was evaluated in a human volunteer study. Twelve apparently healthy adults working in the same environment were randomly assigned into 3 groups with age and gender matched. One group was daily supplemented with a 250 mg tablet containing an aqueous extract of Uncaria tomentosa of C-Med-100, and another group with a 350 mg tablet, for 8 consecutive weeks. DNA repair after induction of DNA damage by a standard dose of hydrogen peroxide was measured 3 times before supplement and 3 times after the supplement for the last 3 weeks of the 8 week-supplement period. There were no drug-related toxic responses to C-Med-100 supplement when judged in terms of clinical symptoms, serum clinical chemistry, whole blood analysis and leukocyte differential counts. There was a statistically significant decrease of DNA damage and a concomitant increase of DNA repair in the supplement groups (250 and 350 mg/day) when compared with non-supplemented controls (p < 0.05). There was also an increased tendency of PHA induced lymphocyte proliferation in the treatment groups. Taken together, this trial has confirmed the earlier results obtained in the rat model when estimating DNA repair enhancement by C-Med-100.

Adult↗

Persistent response to pneumococcal vaccine in individuals supplemented with a novel water soluble extract of Uncaria tomentosa, C-Med-100.

A human intervention study was carried out using male volunteers attending a General Practice Clinic in New York City involving comparison of individuals supplemented with 350 mg x 2 C-Med-100 daily dose for two months with untreated controls for their abilities to respond to a 23 valent pneumococcal vaccine. C-Med-100 is a novel nutraceutical extract from the South American plant Uncaria tomentosa or Cat's Claw which is known to possess immune enhancing and antiinflammatory properties in animals. There were no toxic side effects observed as judged by medical examination, clinical chemistry and blood cell analysis. However, statistically significant immune enhancement for the individuals on C-Med-100 supplement was observed by (i) an elevation in the lymphocyte/neutrophil ratios of peripheral blood and (ii) a reduced decay in the 12 serotype antibody titer responses to pneumococcal vaccination at 5 months.

Adjuvants, Immunologic↗

Enhanced DNA repair, immune function and reduced toxicity of C-MED-100, a novel aqueous extract from Uncaria tomentosa.

Female W/Fu rats were gavaged daily with a water-soluble extract (C-MED-100) of Uncaria tomentosa supplied commercially by CampaMed at the doses of 0, 5, 10, 20, 40 and 80 mg/kg for 8 consecutive weeks. Phytohemagglutinin (PHA) stimulated lymphocyte proliferation was significantly increased in splenocytes of rats treated at the doses of 40 and 80 mg/kg. White blood cells (WBC) from the C-MED-100 treatment groups of 40 and 80 mg/kg for 8 weeks or 160 mg/kg for 4 weeks were significantly elevated compared with controls (P < 0.05). In a human volunteer study, C-MED-100 was given daily at 5 mg/kg for 6 consecutive weeks to four healthy adult males. No toxicity was observed and again, WBC were significantly elevated (P < 0.05) after supplement. Repair of DNA single strand breaks (SSB) and double strand breaks (DSB) 3 h after 12 Gy whole body irradiation of rats were also significantly improved in C-MED-100 treated animals (P < 0.05). The LD50 and MTD of a single oral dose of C-MED-100 in the rat were observed to be greater than 8 g/kg. Although the rats were treated daily with U. tomentosa extracts at the doses of 10-80 mg/kg for 8 weeks or 160 mg/kg for 4 weeks, no acute or chronic toxicity signs were observed symptomatically. In addition, no body weight, food consumption, organ weight and kidney, liver, spleen, and heart pathological changes were found to be associated with C-MED-100 treatment.

Adult↗

Treatment of chemotherapy-induced leukopenia in a rat model with aqueous extract from Uncaria tomentosa.

The Uncaria tomentosa water extracts (C-Med-100) depleted of indole alkaloids (< 0.05%, w/w) have been shown to induce apoptosis and inhibit proliferation in tumor cells in vitro and to enhance DNA repair, mitogenic response and white blood cells in vivo. In this study, the effect of C-Med-100 in the treatment of chemically induced leukopenia was evaluated in a rat model. W/Fu rats were treated first with doxorubicin (DXR) 2 mg/kg x 3 (i.p. injection at 24 hour-intervals) to induce leukopenia. Twenty-four hours after the last DXR treatment, the rats were daily gavaged with C-Med-100 for 16 consecutive days. As a positive control, Neupogen, a granulocyte colony stimulator was also administered by subcutaneous injection at a dose of 5 and 10 microg/ml for 10 consecutive days. The results showed that both C-Med-100 and Neupogen treatment groups recovered significantly sooner (p < 0.05 by Duncan test) than DXR group. However, the recovery by C-Med-100 treatment was a more natural process than Neupogen because all fractions of white blood cells were proportionally increased while Neupogen mainly elevated the neutrophil cells. These results were also confirmed by microscopic examination of the blood smears. The mechanism of the C-Med-100 effect on WBC is not known but other data showing enhanced effects on DNA repair and immune cell proliferative response support a general immune enhancement.

Administration, Oral↗

N-substituted benzamides inhibit NFkappaB activation and induce apoptosis by separate mechanisms.

Benzamides have been in clinical use for many years in treatment against various disorders. A recent application is that as a sensitizer for radio- or chemotherapies. We have here analysed the mechanism of action of N-substituted benzamides using an in vitro system. We found that while procainamide was biologically inert in our system, the addition of a chloride in the 3' position of the benzamide ring created a compound (declopramide) that induced rapid apoptosis. Furthermore, declopramide also inhibited NFkappaB activation by inhibition of IkappaBbeta breakdown. An acetylated variant of declopramide, N-acetyl declopramide, showed no effect with regard to rapid apoptosis induction but was a potent inhibitor of NFkappaB activation. In fact, the addition of an acetyl group to procainamide in the 4' position was sufficient to convert this biologically inactive substance to a potent inhibitor of NFkappaB activation. These findings suggest two potential mechanisms, induction of early apoptosis and inhibition of NFkappaB mediated salvage from apoptosis, for the biological effect of N-substituted benzamides as radio- and chemo-sensitizers. In addition it suggests that N-substituted benzamides are potential candidates for the development of anti-inflammatory compounds using NFkappaB as a drug target.

Apoptosis↗

Pharmacokinetics and central nervous system toxicity of declopramide (3-chloroprocainamide) in rats and mice.

Declopramide (3-chloroprocainamide) has been identified in previous studies as a representative of a new class of chemosensitizers. In this study, the toxicity and pharmacokinetics of declopramide have been investigated and compared with a structural analog, metoclopramide (MCA). Declopramide has not induced central nervous system (CNS)-related side effects in rats at doses up to 200 mg/kg, whereas MCA does at 12.5 mg/kg. In addition, declopramide did not bind to dopamine D2 receptors in subcellular preparations at doses up to 100 microM, whereas MCA showed affinity at 1 microM. Declopramide bound with affinity to 5-hydroxytryptamine3 receptors which are important in controlling vomiting. In contrast to MCA, declopramide has a rapid clearance from serum, a lower tissue concentration (about 15-fold lower than MCA) and a lower oral bioavailability (about 6-fold lower than MCA). However, declopramide was shown in vitro to possess a higher tumor cell absorption rate. One of the main metabolites of declopramide was identified as N-acetyl declopramide. Taken together, these data suggest that the clinical development of declopramide as a sensitizer of radio- and chemotherapies is an improvement over MCA, because it can be administered in a high dose and is devoid of CNS side effects.

Administration, Oral↗

Newly discovered anti-inflammatory properties of the benzamides and nicotinamides.

Our laboratory has concentrated on the possible regulation the benzamides and nicotinamides may have on the processes of DNA repair and apoptosis. Recent reports have suggested that both apoptosis and inflammation are regulated by the transcription factor NF-kappaB. We have initiated studies regarding the hypothesis that the benzamides and nicotinamides could inhibit the production of tumor necrosis factor alpha (TNFalpha) and the inflammatory response as well as induce apoptosis via inhibition of NF-kappaB. Our data have shown that nicotinamide and two N-substituted benzamides, metoclopramide (MCA) and 3-chloroprocainamide (3-CPA), gave dose dependent inhibition of lipopolysacharide induced TNFalpha in the mouse within the dose range of 10-500 mg/kg. Moreover, lung edema was prevented in the rat by 3 x 50 mg/kg doses of 3-CPA or MCA, and 100-200 microM doses of MCA could also inhibit NF-kappaB in Hela cells. Taken together these data strongly support the notion that benzamides and nicotinamides have potent anti-inflammatory and antitumor properties, because their primary mechanism of action is regulated by inhibition at the gene transcription level of NF-kappaB, which in turn inhibits TNFalpha and induces apoptosis.

Animals↗

Zinc is an essential cofactor for recognition of the DNA binding domain of poly(ADP-ribose) polymerase by antibodies in autoimmune rheumatic and bowel diseases.

OBJECTIVE: To characterize autoantibody response to poly(ADP-ribose) polymerase (PARP) and to assess the significance of autoantibodies to the 2 zinc fingers of this enzyme in patients with autoimmune rheumatic and bowel diseases. METHODS: The specificity of antienzyme autoantibodies was established by dot immunoassay with recombinant human PARP and by enzyme-linked immunosorbent assay using the recombinant N-terminal fragment containing the DNA binding domain of PARP, the recombinant C-terminal catalytic domain (40-kd fragment), a peptide containing the nuclear localization signal (NLS) of PARP, 2 synthetic peptides (and mutated peptides) corresponding to zinc-finger motifs F1 and F2 that are present in the DNA binding domain, zinc fingers from other self antigens (e.g., peptides from Ro60, Ro52, and U1C proteins), and poly(ADP-ribose). Sera from patients with autoimmune rheumatic and bowel diseases were tested, as were affinity-purified antibodies. Histocompatibility typing of systemic lupus erythematosus (SLE) patients was performed by serology. RESULTS: Antibodies from the patient sera reacted only weakly with the recombinant N- and C-terminal domains and with the NLS peptide. In contrast, the 2 synthetic peptides corresponding to zinc-finger motifs F1 and F2 represented immunodominant targets for IgG antibodies from patients with SLE, mixed connective tissue disease (MCTD), Crohn's disease, and ulcerative colitis. The sera from patients with SLE and MCTD showed much weaker reactivity with mutant peptides F1 and F2, which contain mutations at the cysteine residues involved in zinc coordination. F1/F2 antibodies did not cross-react with zinc fingers from other self proteins. No correlation was found between the presence of F1/F2 autoantibodies in SLE sera and the presence of other autoantibodies typical of this disease (e.g., anti-double-stranded DNA and poly[ADP-ribose] antibodies). The presence of F2 antibodies in the serum of SLE patients was negatively associated with HLA-DR6. CONCLUSION: An autoimmune response to PARP is potentially important because this enzyme is involved in DNA repair and is rapidly cleaved during the "execution phase" of apoptosis. The high prevalence in certain autoimmune rheumatic and bowel diseases of antibodies to F1 and F2, which are directly involved in this process, is further evidence implicating involvement of the DNA repair system in chronic inflammatory diseases.

Amino Acid Sequence↗

Multiple mechanisms of action of the benzamides and nicotinamides as sensitizers of radiotherapy: opportunities for drug design.

The benzamides and nicotinamides are a well-known class of drugs that contain many analogs having radio- and chemosensitizing properties. This study reports on a structural analysis in order to explain the chemical features important to their mechanisms of action. In general, N-substituted analogs are distinguished from the non-N-substituted analogs because they (i) are susceptible to radiolysis, (ii) induce cytotoxicity by apoptosis but not necrosis, (iii) inhibit cell proliferation, (iv) activate poly adenosine diphosphate ribosyl transferase (poly ADPRT), and (v) have a much-reduced effect on microregional tumor blood perfusion. It was concluded that the mechanism of action of N-substituted analogs is shifted from primary effects on tumor vascularization as is seen with the non-N-substituted analogs to one where radiosensitization can be explained by selective induction of apoptosis via radiolysis and accumulation of DNA damage. This knowledge may be useful in the design of drugs possessing multiple mechanisms of radiosensitizing action.

Animals↗

DNA repair enhancement by a combined supplement of carotenoids, nicotinamide, and zinc.

Four volunteers were involved for 5 weeks of a baseline period, followed by 7 weeks of a combined supplementation of nicotinamide, zinc, and carotenoids (Nicoplex). Blood sampling and bioassays were carried out every week during the evaluation period. The supplementation of Nicoplex resulted in statistically significant increased resistance to DNA single-strand breaks induced by H2O2 (DNA retained on filter % from 46.7 +/- 1.9 to 59.4 +/- 4.3; p < 0.01), increased DNA repair 60 min after induction of damage (DNA retained on filter % from 74.6 +/- 4.8 to 88.3 +/- 4.2; p < 0.01), elevated poly (ADP-ribose) polymerase (PARP) activity (p < 0.05), and an increased proliferative response to phytohemagglutinin (PHA) (p < 0.05) when compared with the levels before supplementation. However, when the same subjects were supplemented with nicotinamide, zinc, and carotenoids together with another 17 nutrients or minerals, there were no changes in DNA damage, DNA repair, or proliferative response to PHA. Through the use of a rat model, DNA repair of splenocytes 3 h after 12 Gy whole-body irradiation was significantly enhanced in rats supplemented with Nicoplex for 6 weeks (p < 0.05) and 8 weeks (p < 0.01). Comparison of Nicoplex and its components administered separately revealed that there was an additive effect on DNA repair for both single- and double-strand breaks (both p < 0.05). On the basis of the results, it is hypothesized that the enhanced effect of combined supplement of nicotinamide, zinc, and carotenoids on DNA repair depends on their diversified mechanisms of action while multinutrient supplementation may compromise the effects by inhibitory interactions including uptake and absorption.

Adult↗

Quality control program for storage of biologically banked blood specimens in the Malmö Diet and Cancer Study.

A biological bank has been developed to extend the biochemical and molecular research base for a prospective study on diet and cancer in the city of Malmo, Sweden. The study entered individuals 45-69 years of age, of which 30,382 individuals (45%) participated. Each individual entering the bank has stored samples of viable mononuclear leukocytes (MNLs; -140 degrees C) and granulocytes (GRANs; -80 degrees C) or buffy coats (-140 degrees C), erythrocytes (-80 degrees C), and plasma/serum (-80 degrees C). The bioassays developed to monitor the quality of storage conditions were: (a) viability and growth response to phytohemagglutinin for MNLs; (b) DNA strand breakage for GRANs; (c) NAD content for erythrocytes; and (d) thiol status for plasma/serum. The yield, purity, and storage conditions were all quality controlled, and the samples were determined to be of high standard after 137-190 weeks of storage. No differences in yield and purity were found in samples banked by different laboratory technicians. Growth responses of MNLs were severely reduced (90%) after 40 weeks of storage, which justified switching from the storage of purified MNLs and GRANs to the more cost-effective banking of buffy coats. We conclude that the quality of the banked material, based on the biochemical analysis done, indicate that the storage conditions are optimal at least up to 3.5 years, except for the growth response of MNLs.

Blood Banks↗

Feasibility and quality of biological banking of human normal and tumor tissue specimens as sources of DNA for the Malmö Diet and Cancer Study.

Human tumor and normal tissue specimens, which were collected from autopsy material 1-6 days postmortem, were compared with similar tissue specimens collected within 2 h after surgical resection and transport to the pathology department. The end point criteria used to evaluate the quality of the specimens for biological banking purposes were the extractability and yield of high molecular weight DNA and UV absorption ratios at 260:280 after collection and immediate storage of the specimens at -80 degrees C. The data demonstrated that autopsy material was a quality source of DNA, although of not such high quality as surgical biopsy specimens <2 h after resection. The advantages of using autopsy material to supplement surgical specimen collection sent to pathology, as opposed to using specimen collection at surgery wards or formalin-fixed material, as sources of DNA are: (a) large amounts of tumor and normal tissues from a variety of organ sites can be obtained without regard to the patient's health status; (b) a higher percentage of retrieval of incident cases of cancer in prospective designed trials is more likely to be achieved; and (c) the extractable DNA is of sufficiently high enough quality to permit direct analyses by molecular hybridization and sequence methodologies.

Autopsy↗

Whole-body hyperthermia and ADPRT inhibition in experimental treatment of brain tumors.

Malignant primary brain tumors have hitherto been incurable. One reason for this may be the migrating tumor cells that spread into the surrounding normal brain, creating the basis for inevitable recurrences. Therefore, local therapy may have a temporary effect, but for a cure, the treatment must reach all the tumor cells. Whole-body hyperthermia (WBH) is such a general treatment, which we have studied in a rat glioma model. Cells of the RG2 rat glioma cell line were inoculated in the right caudate nucleus of Fischer-344 rats, which were then either controls or treated with WBH, induced by a radiant heat device for 4-5 sessions of 30 min at body temperature 42 degrees C (rectal), and/or nicotinamide (NAM), an effective radiosensitizer in animal tumor models and an inhibitor of ADPRT (poly adenosine diphosphate ribosyl transferase), a chromatin-bound enzyme suggested to be important in the DNA repair system. We have shown that WBH 42 degrees C alone, or in combination with NAM, has no effect upon tumor growth if a larger number of RG2 cells (5,000) are inoculated. If only 1,000 cells are inoculated, a significant inhibitory effect (p < 0.05) is observed on tumor growth as compared to the untreated control animals. Thus, WBH is feasible, and in some circumstances effective, in a rat glioma model. WBH in combination with other therapies influencing cell metabolism may be of value in future postoperative treatment of human malignant brain tumors.

Animals↗

Neutral metoclopramide sensitizes cytotoxicity induced by ionizing radiation in SCID mice xenografted with a human brain astrocytoma.

A formulation of metoclopramide (MCA) conformationally altered by neutralization of pH (nMCA, Neu-Sensamide) has been shown to have the same efficacy of enhancing the cytotoxicity of a single dose of 1 Gy radiation as acidic formulations (e.g., Primperan, Sensamide) in a human lung adenocarcinoma (H2981) xenografted into SCID mice. In the present study, 2 x 1 Gy radiation was combined with 2 x 2 mg nMCA/kg body weight injected 2 hr before radiation treatment for evaluation of radiosensitization in SCID mice xenografted with a human brain astrocytoma (T24). Given in this treatment schedule, nMCA alone at 2 mg/kg showed no cytotoxic effect on tumor growth in vivo. When combined with 2 x 1 Gy of radiation, however, the cytotoxicity was significantly increased as measured by tumor growth delay over the radiation-only-treated group. Furthermore, nMCA was absorbed into brains of mice and rats as efficiently as acidic MCA (aMCA) when analyzed 45 min after i.m. injection by high-performance liquid chromatography.

Animals↗

Comparative central nervous system effects and pharmacokinetics of neu-metoclopramide and metoclopramide in healthy volunteers.

Metoclopramide, a drug used for the relief of nausea and emesis, is currently under development as a radio- and chemosensitizing agent. Its usefulness in high doses, however, is limited by its central nervous system side effects. Neu-metoclopramide (Neu-Sensamide), a novel, concentrated, phosphate-buffered, pH-adjusted (pH = 6.5-7.0) formulation of metoclopramide, has been shown to have an improved side-effect profile in animal studies. The present double-blind, four-way crossover study compared the central nervous system effects and pharmacokinetics of neu-metoclopramide (intravenously and intramuscularly at 1.8 mg/kg) with intravenous metoclopramide and intramuscular placebo in 19 healthy male volunteers. Eight participants withdrew from the study, one because of noncompliance and seven because of adverse events. A total of 28 central nervous system events were observed with intravenous metoclopramide administration, whereas 16, 15, and 6 such events were attributed to intravenous neu-metoclopramide, intramuscular neu-metoclopramide, and placebo, respectively. Extra-pyramidal effects occurred on 10 occasions: 7 after intravenous metoclopramide, 2 after intravenous neu-metoclopramide, and 1 after intramuscular neu-metoclopramide. No significant differences were observed in the pharmacokinetic profiles of the three formulations of metoclopramide. It may be speculated, therefore, that the molecular conformational changes inherent to neu-metoclopramide result in a reduced side-effect profile compared with conventional metoclopramide formulations.

Adult↗