Vitamin D3 seems more appropriate than D2 to sustain adequate levels of 25OHD: a pharmacokinetic approach.

BACKGROUND/OBJECTIVES: The superiority of cholecalciferol (D3) over ergocalciferol (D2) in sustaining serum 25-hydroxy vitamin D (25OHD) levels is controversial. To compare D2 with D3 we performed a single-blind, placebo-controlled randomized trial spanning 11 weeks. SUBJECTS/METHODS: Healthy volunteers (n=33, aged 33.4±6 years) were divided into three groups (n=11, each): D2, D3 and placebo. Treatment started with a loading dose (100,000 IU) followed by 4800 IU/day (d) between d7 and d20 and follow-up until d77. Serum samples were obtained at baseline and at days 3, 7, 14, 21, 35, 49, 63 and 77. RESULTS: Baseline 25OHD values in the D2 group were lower than those in the D3 and placebo groups (P<0.01). Placebo 25OHD levels never changed. As after the loading dose both D2 and D3 groups had reached similar 25OHD levels, we tested equivalence of the area under the concentration × time curve (AUC) between d7 and d77. The AUC was 28.6% higher for D3 compared with D2, and both were higher with respect to placebo. At d77, D2 25OHD levels were higher than those at baseline, but similar to placebo; both were lower than D3 (P<0.04). According to raw data, the elimination half-life of 25OHD was 84 and 111 days under D2 and D3 supplementation, respectively; after subtracting the placebo values, the corresponding figures were 33 and 82 days. CONCLUSIONS: D2 and D3 were equally effective in elevating 25OHD levels after a loading dose. In the long term, D3 seems more appropriate for sustaining 25OHD, which could be relevant for classic and non-classic effects of vitamin D.

Chronic inflammatory demyelinating polyradiculoneuropathy in a patient with systemic lupus erythematosus and good outcome with rituximab treatment.

A patient with chronic inflammatory demyelinating polyradiculoneuropathy and systemic lupus erythematosus arising after rubella vaccination was initially treated with plasmapheresis, corticosteroids and intravenous immunoglobulins, with partial response. After shift to rituximab, most clinical symptoms improved markedly, emphasizing the possible role of this monoclonal antibody in conventional therapy-resistant cases.

Immunogenicity of an interferon-beta1a product.

In order to determine whether Blastoferon®, a biosimilar interferon (IFN)- beta 1a formulation, shares epitopes with other known IFN-beta products, a series of neutralization bioassays were performed with a set of well-characterized anti-IFN- beta monoclonal antibodies and human sera (World Health Organization Reference Reagents). The bioassay was the interferon-induced inhibition of virus cytopathic effect on human cells in culture (EMC virus and A-549 cells). Computer-calculated results were reported as Tenfold Reduction Units (TRU)/ml. To further assess Blastoferon® immunogenicity, in vivo production of anti-IFN beta antibodies was determined in sera of patients included in the pharmacovigilance plan of Blastoferon® by the level of IFN- beta 1a binding antibodies (by enzyme immunoassay -EIA) and neutralizing antibodies (in the Wish-VSV system). The highly characterized neutralizing monoclonal antibodies A1 and A5 that bind to specific regions of the IFN- beta molecule reacted positively with the three beta 1a IFNs: Blastoferon®, Rebif®, and the IFN- beta WHO Second International Standard 00/572. As expected, the non-neutralizing monoclonal antibodies B4 and B7 did not neutralize any of the IFN- beta preparations. The commercially available monoclonal antibody B-02 reacted essentially equally with Rebif® and Blastoferon®. The WHO Reference Reagent human serum anti-IFN- beta polyclonal antibody neutralized all the IFN- beta products, whereas the WHO Reference Reagent human serum anti-IFN-alpha polyclonal antibody G037-501-572 appropriately failed to react with any of the IFN- beta products. On the basis of in vitro reactivity with known, well-characterized monoclonal and polyclonal antibody preparations, Blastoferon® shares immunological determinants with other human interferon- beta products, especially IFN- beta 1a. In vivo antibodies were detected by EIA in 72.9% of 37 chronically treated multiple sclerosis patients, whereas neutralizing antibodies were found in 8.1% of them. Blastoferon® appears to have immunological characteristics comparable to other IFN- beta 1a products.

Relative bioavailability of two drug products of somatropin obtained from either the milk of transgenic cows or bacterial culture.

BACKGROUND: Our objective was to assess the relative bioavailability of the first somatropin produced in transgenic cloned cows that carry the human growth hormone (GH) gene (Biohormon) and somatropin produced in Escherichia coli culture (HHT), the procedure most frequently used for the commercial production of the hormone. METHODS: Upon approval by an independent ethics committee and the National Regulatory Agency of Argentina, we compared the time-concentration profiles of somatropin in 24 healthy volunteers, in a randomized, 2-period, 2-sequence crossover design after inhibition of endogenous GH secretion with lanreotide, a long-acting somastostatin analogue. After the subcutaneous administration of 1.33 mg of each formulation, serum somatropin was analyzed by chemiluminescent immunoassay and IGF-I by immunoradiometric assay. Safety was assessed by clinical and laboratory parameters. Pharmacokinetic parameters were calculated with Win Nonlin 5.2 using a non-compartmental model and bioequivalence was assessed. RESULTS: The test/reference ratios of AUC, AUC(last) and C(max) were 106.4 (90% CI = 100.2-112.9), 105.3 (90% CI = 99.1-111.8) and 105.49 (90% CI = 92.6-120.1), respectively. No serious adverse events were reported and no GH antibodies were detected. CONCLUSION: This study demonstrates that a single dose of Biohormon, the first product with somatropin obtained from milk of transgenic mammals, is bioequivalent to the reference product HHT according to standard criteria.

Whole genome analysis of the action of interferon-beta.

OBJECTIVES: To characterize the IFNbeta1a-regulated gene expression on leukocytes of Multiple Sclerosis (MS) patients using microarrays with whole human genome representation. METHODS: Genes differentially expressed by interferon-beta were identified by a microarray in vitro study performed in leukocytes obtained from 5 MS relapsing-remitting patients. RESULTS: Following the culture of peripheral blood mononuclear cells from MS relapsing-remitting patients for 24 hs with IFNbeta1a, the expression of 868 genes was modified: 545 increased (including CXCL11, CCL8, INDO, IFI27, CFB, CXCL10 and IFIT1) and 323 diminished (including RBP7, SEPT5, RNF8, ADORA2B and FOS). CONCLUSIONS: Since many of them were previously recognized as involved in MS pathogenesis, the IFNbeta1a mechanism of action could imply a compensatory regulation of systems deregulated in MS.

Two recombinant human interferon-beta 1a pharmaceutical preparations produce a similar transcriptional response determined using whole genome microarray analysis.

OBJECTIVES: Recombinant human interferon-beta (IFN-b) is a well-established treatment for multiple sclerosis (MS). The regulatory process for marketing authorization of biosimilars is currently under debate in certain countries. In the EU, EMEA has clearly defined the process including overarching and product-specific guidelines, which includes clinical testing. Biosimilarity needs to be based on comparability criteria, including at least molecular characterization, biological activity relevant for the therapeutic effect and relative bioavailability ("bioequivalence"). In the case of such complex diseases as MS, where the effect of treatment is not so directly measurable, in vitro tools can provide additional data to support comparability. Genomic microarrays assays might be useful to compare multisource biopharmaceuticals. The aim of the present study was to compare the pharmacodynamic genomic effects (in terms of transcriptional regulation) of two recombinant human IFN-I(2)1a preparations on lymphocytes of multiple sclerosis patients using a whole genome microarray assay. METHODS: We performed an ex vivo whole genome expression profiling of the effect of two preparations of IFN-I(2)1a on non-adherent mononuclears from five relapsing-remitting MS patients analyzing microarrays (CodeLink Human Whole Genome). Patients blood was drawn, PBMCs isolated and cultured in three different conditions: culture medium (control), 1,000 U/ml of IFN-I(2)1a (BLA- (STOFERON, Bio Sidus) and 1,000 U/ml of IFN-I(2)1a (REBIF, Serono) RNA was purified from non-adherent cells (mostly lymphocytes), amplified and hybridized. Raw data were generated by CodeLink proprietary software. Data normalization, quality control and analysis of differential gene expression between treatments were done using linear model for microarray data. Functional annotation analysis of IFN-I(2)1a MS treatment transcription was done using DAVID. RESULTS: Out of the approximately 45,000 human sequences examined, no evidence of differential regulation was found when both treatments were compared (minimum adjusted p-value > 0.999). The IFN-I(2)1a effect differentially regulated the expression of 868 genes. The expression of standard markers such as GTP cyclohidrolase, MxA, and OAS isoenzymes A and B changed as a consequence of the action of IFN-I(2)1a. CONCLUSIONS: This exhaustive and highly sensitive assay did not show differences in the genomic expression profile of these two products under the assayed experimental conditions. These results suggest that this technology might be useful for the initial comparison of biosimilars, being part of a comprehensive comparability program that includes clinical testing.

Fatal portal thrombosis after laparoscopic Nissen fundoplication.

Portal and mesenteric vein thrombosis is a very uncommon complication of laparoscopic surgery, especially after anti-reflux procedures. We report the case of a twenty-year-old man with a history of alcohol and cocaine consumption. A Nissen fundoplication was performed. The patient received a single 20-mg dose of enoxaparin (Clexane, Aventis Pharma, Spain) two hours before surgery for antithrombotic prophylaxis. On the seventh postoperative day the patient had a portal and mesenteric venous thrombosis, which was confirmed at laparotomy, with both extensive small-intestine necrosis and partial colon necrosis. Despite anticoagulant therapy, the patient died 24 hours later. Surgical findings were confirmed at necropsy. Portal and mesenteric venous thrombosis is an uncommon but severe and even fatal complication after laparoscopic anti-reflux surgery. When other pro-thrombotic, predisposing conditions such as laparoscopic surgery and cocaine consumption are present, the usual prophylactic doses of low molecular weight heparin might not be sufficient to protect against this life-threatening complication.

Detection of interleukin 10 in cerebrospinal fluid of patients with subacute sclerosing panencephalitis.

Subacute sclerosing panencephalitis (SSPE) is a neurodegenerative disorder due to persistent measles virus infection, with high level of measles-specific antibodies in cerebrospinal fluid (CSF). To analyze whether such response arises from a TH2-biased response, the authors determined TH1 (interferon [IFN]-gamma) and TH2 (interleukin [IL]-4 and IL-10) cytokines in CSF, taken at diagnosis, of eight SSPE patients (median age, 57.5 month, range 42 to 76 months). All patients presented IL-10 (median 29.3 pg/ml, range 4.3 to 162 pg/ml), but not IL-4 (<10 pg/ml); only one case showed IFN-gamma (162 pg/ml). These results are consistent with a TH2 bias or with a local, anti-inflammatory or neuroprotective mechanism involving IL-10.

Vaccines in Argentina: a regulatory view.

In Argentina, vaccines for immuno-preventable diseases are regulated by the national regulatory agency, the Administración Nacional de Medicamentos, Alimentos y Tecnología Médica (the National Administration of Drugs, Food and Medical Devices, or ANMAT) created in 1992 to ensure efficacy and safety of drugs, food and medical devices available in the country, according to Law 16,463 and Decree 150/92. ANMAT has licensed 84 out of 157 vaccines registered in Argentina. Since 1994, ANMAT evaluated, approved and inspected 20 clinical trials with vaccines (1.8% of the 1062 trials approved by the agency since that time). The National System of Pharmaco-vigilance has received 318 communications of eventual adverse post-vaccination events (0.3% of the total). In addition, ANMAT provides support to the National Immunisation Programme. The current procedure is to follow international guidelines in the field, to be prepared for new, rapidly changing scenarios.

Effect of ovaric hyper-stimulation on blood lymphocyte subpopulations, cytokines, leptin and nitrite among patients with unexplained infertility.

PROBLEM: To determine the effect of ovarian stimulation on TH1, TH2 and natural killer (NK) lymphocytes, plasma cytokines, leptin and nitrite levels. METHODS: Women with reproductive failure were studied during the implantation window, at baseline (n = 18) and under ovarian stimulation (gonadotropins + progesterone, n = 6). CONTROLS: eight fertile women. Lymphocyte subpopulations and NK function were determined by flow cytometry. Interleukin-2 (IL-2), IL-4, IL-10, IFN-gamma, TNF-alpha, TGF-beta1 and leptin were measured by enzyme immunoassay (EIA); nitrite by the Griess reaction. RESULTS: At baseline, patients had higher values of NK effectors, NK activity and plasma IFN-gamma and IL-2 than controls. Conversely, TGF-beta1 values were lower. Hormones induced leukocytosis. Under stimulation, THI CD4+ cells, NK effectors and function and plasma IFN-gamma and IL-2 decreased, while transforming growth factor (TGF)-beta1 increased. Other variables did not change. CONCLUSION: The abnormal distribution of leukocytes, high TH1 cytokines and a low TGF-beta1 associated with reproductive failure, respond to ovarian stimulation, achieving total or partial normalization.

High serum interleukin-10 and tumor necrosis factor alpha levels in chronic paracoccidioidomycosis.

In patients with chronic paracoccidioidomycosis (n = 10), levels of tumor necrosis factor alpha, interleukin-10, and interleukin-2 in serum, measured by enzyme-linked immunosorbent assay (in picograms per milliliter, as mean +/- standard error of the mean), were higher than in normal controls (n = 8): 186 +/- 40 versus 40 +/- 7 (P < 0.05), 203 +/- 95 versus 20 +/- 8 (P = 0.001), and 96.3 +/- 78.57 versus 1.19 +/- 1.19 (P = 0.045), respectively. Gamma interferon and interleukin-4 levels were similar in patients and controls.

Neutrophil respiratory burst activity and pro- and anti-inflammatory cytokines in AAA surgery: conventional versus endoluminal treatment.

PURPOSE: To examine the inflammatory impact of endovascular and conventional surgery of abdominal aortic aneurysm (AAA) as assessed by the activation of neutrophils and serum levels of pro- and anti-inflammatory cytokines. METHODS: Twenty-four patients undergoing AAA exclusion were treated with either endovascular (n = 14) or conventional (n=10) techniques. Clinical and hematological data, respiratory burst of neutrophils, and the expression of adhesion and activation molecules (CD18, CD11b, CD69, and HLA-DR) were analyzed by flow cytometry. The enzyme-linked immunosorbent assay technique was used to measure proinflammatory cytokine interleukin (IL)-8 and anti-inflammatory cytokines IL-1 receptor antagonist (IL-1RA) and transforming growth factor beta1 (TGF-beta1). RESULTS: All patients, most of whom had normal cytokine values before surgery, were successfully treated. No significant changes were found in surface antigens. Basal respiratory burst was quite heterogeneous; in all cases respiratory burst activity decreased after surgery and remained low throughout the observation period. Despite marked interpatient differences, IL-1RA and IL-8 increased after surgery, whereas TGF-beta1 decreased, although the variation achieved statistical significance only in the conventional group. Elevated IL-1RA returned to normal within 48 hours in the endoluminal group, whereas the level remained high in the conventional group in the last sample. CONCLUSIONS: Despite heterogeneity before surgery, the respiratory burst decreased for most of the patients regardless of the approach, and both techniques increased IL-1RA. Although both procedures seemed to decrease TGF-beta1, the difference was significant only with the conventional approach. IL-1RA levels fell toward basal values quicker in the endograft patients, suggesting that the endoluminal approach was less aggressive.

In vivo effect of snake phospholipase A2 (crotoxin+cardiotoxin) on serum IL-1alpha, TNF-alpha and IL-1ra level in humans.

VRCTC-310-Onco (crotoxin, a secretory phospholipase A2+cardiotoxin) is under development as an anti-neoplastic agent. Pro-inflammatory cytokines TNF-alpha and interleukin 1 alpha (IL-1alpha) and anti-inflammatory cytokine IL-1 receptor antagonist (IL-1ra) were measured with commercial ELISA kits in sera corresponding to 23 cycles with doses between 0.0025 and 0.023 microg/kg body weight, obtained during the phase I trial of VRCTC-310-Onco. Neither serum TNF-alpha nor IL-1alpha did change significantly after VRCTC-310-Onco. Basal IL-1ra was 794 +/- 97 pg/ml, by 3 h it was similar, 651 +/- 99 pg/ml and at 24 h p.i. it increased to 1197 +/- 122 pg/ml (P<0.001). The increase was dose-dependent. The addition of dexamethasone (required to reduce pain with the highest doses) inhibited IL-1alpha and enhanced the induction of IL-1ra by VRCTC-310-Onco. Summing up, in vivo, in humans, in the dose range tested, VRCTC-310-Onco induces IL-1ra, and does not consistently modify IL-1alpha or TNF-alpha serum levels.

Serum transforming growth factor-beta1 levels increase in response to successful anti-inflammatory therapy in ulcerative colitis.

OBJECTIVE: To investigate serum levels of transforming growth factor-beta1 and interferon-gamma in active ulcerative colitis and to assess changes during treatment. METHODS: We prospectively evaluated serum from 25 patients with untreated active ulcerative colitis and 19 healthy controls. Disease activity score (DAI), serum transforming growth factor-beta1 and interferon-gamma levels were measured at baseline and after 7 days of conventional treatment. Disease activity score and transforming growth factor-beta1 were also assessed at 42 days. RESULTS: Baseline transforming growth factor-beta1 levels were significantly higher in patients than in controls (P < 0.02). On the 7th day, transforming growth factor-beta1 levels increased only in patients who responded (P < 0. 01); variations in transforming growth factor-beta1 levels and disease activity score were inversely correlated (r=- 0.72, P < 0. 001). At day 42, serum transforming growth factor-beta1 decreased significantly compared with the 7th day (P < 0.05). While in controls, interferon-gamma was undetectable; untreated patients had higher, widely variable, levels. At day 7, responders had higher interferon-gamma values than unresponsive cases. Variations in interferon-gamma correlated moderately with changes in transforming growth factor-beta1 (r=0.53, P < 0.05). Cytokine response did not depend upon the type of treatment. CONCLUSIONS: Both transforming growth factor-beta1 and interferon-gamma may play a role in the injury-repair process in active ulcerative colitis. Variations in circulating transforming growth factor-beta1 levels in the first week of treatment seem to be related to the therapeutic response.

One-month safety study of intraperitoneal VRCTC-310-Onco (crotoxin + cardiotoxin) in rats.

To evaluate the toxicity of VRCTC-310-Onco (Crotalus durissus terrificus crotoxin + cardiotoxin from Naja naja atra), 10 Sprague-Dawley rats were implanted with intraperitoneal slow-release devices and subjected to treatment with 0.5 microgram/g body weight/d for 14 days. Biochemical evidence at days 7 and 14 showed blood, muscular, renal and metabolic disturbance, mostly reversed by day 28. No significant changes were found in necropsy. The limited toxicity of i.p. VRCTC-310-Onco in rats deserves further study.

Lymphocyte subsets and cytokines in adenoviral infection in children.

To determine the distribution of major blood lymphocyte subsets we evaluated blood lymphocytes by flow cytometry in adenovirus-infected infants aged 30-730 d. In addition, interleukin-1-receptor antagonist, interleukin-10 and transforming growth factor-beta1 were measured in serum by enzyme-linked immunosorbent assay. According to clinical parameters, mechanical ventilation and outcome, infections were classified as moderate (n = 15), severe (n = 11) and fatal (n = 12). Controls were 13 healthy children. In severe and fatal infection, T cells (CD5+/CD19-), NK effectors (CD16+), CD4+ T subset and B1 subset of B lymphocytes (CD5+/CD19+) were all significantly decreased. CD8+ cells were decreased in severe but not fatal cases. There was no difference in serum values of interleukin-10; however, fatal cases had high interleukin 1-receptor antagonist values. Interestingly, patients with moderate infection showed significantly increased values of transforming growth factor-beta1. These results demonstrate that life-threatening adenoviral infection is associated with marked abnormalities in blood lymphocyte and cytokine profile.