Activation of nucleotide-binding oligomerization domain 2 by muramyl dipeptide negatively regulates Toll-like receptor 9-mediated colonic inflammation through the induction of deubiquitinating enzyme A expression.

Mutations in nucleotide-binding oligomerization domain 2 (NOD2) are associated with Crohn's disease (CD). Although NOD2 activation contributes to the maintenance of intestinal homeostasis through the negative regulation of pro-inflammatory cytokine responses mediated by Toll-like receptors (TLRs), the effects of NOD2 activation on interferon (IFN)-α responses induced by TLR9 have been poorly defined. To explore the cross-talk between NOD2 and TLR9, human monocytes or dendritic cells (DCs) were stimulated with NOD2 and/or TLR9 ligands to measure IFN-α production. The severity of dextran sodium sulfate (DSS)-induced colitis was compared in mice treated with NOD2 and/or TLR9 ligands. Expression of IFN-α and IFN-stimulated genes (ISGs) was examined in the colonic mucosa of patients with inflammatory bowel disease (IBD). NOD2 activation reduced TLR9-induced IFN-α production by monocytes and DCs in a deubiquitinating enzyme A (DUBA)-dependent manner. Activation of DUBA induced by the co-stimulation of TLR9 and NOD2 inhibited Lys63-linked polyubiquitination of TRAF3 and suppressed TLR9-mediated IFN-α production. NOD2 activation in hematopoietic cells protected mice from TLR9-induced exacerbation of DSS-induced colitis by down-regulating IFN-α responses and up-regulating DUBA expression. Colonic mucosa of patients with active and remitted IBD phases was characterized by the enhanced and reduced expression of ISGs, respectively. Expression levels of IFN-α and IL-6 positively correlated in the active colonic mucosa of patients with ulcerative colitis and CD, whereas DUBA expression inversely correlated with that of IFN-α in patients with CD. Collectively, these data suggest that DUBA-dependent negative effect of NOD2 on TLR9-mediated IFN-α responses contributes to the maintenance of intestinal homeostasis.

Sensing of commensal organisms by the intracellular sensor NOD1 mediates experimental pancreatitis.

The intracellular sensor NOD1 has important host-defense functions relating to a variety of pathogens. Here, we showed that this molecule also participates in the induction of a noninfectious pancreatitis via its response to commensal organisms. Pancreatitis induced by high-dose cerulein (a cholecystokinin receptor agonist) administration depends on NOD1 stimulation by gut microflora. To analyze this NOD1 activity, we induced pancreatitis by simultaneous administration of a low dose of cerulein (that does not itself induce pancreatitis) and FK156, an activator of NOD1 that mimics the effect of gut bacteria that have breached the mucosal barrier. The pancreatitis was dependent on acinar cell production of the chemokine MCP-1 and the intrapancreatic influx of CCR2(+) inflammatory cells. Moreover, MCP-1 production involved activation of the transcription factors NF-κB and STAT3, each requiring complementary NOD1 and cerulein signaling. These studies indicate that gut commensals enable noninfectious pancreatic inflammation via NOD1 signaling in pancreatic acinar cells.

The Efficiency and Toxicity of Mifamurtide in Childhood Osteosarcoma.

The aim of the present study was to evaluate the efficiency and side effects of mifamurtide in childhood osteosarcoma (OS). In total, 477 doses of 2 mg/m intravenous (IV) mifamurtide, along with paracetamol as a premedication, were given to 15 patients with primary nonmetastatic OS after complete surgical resection and to 3 patients with progressive OS. The most common side effects encountered in the patients were chills and fever (17/18). These reactions were observed in 4 patients during the administration of each dose, in a single patient during the last administration, and in the remaining 12 patients during the first or initial 2 administrations. Headache, myalgia, and arthralgia were observed in 2 patients during each infusion. Headache was observed in 1 patient with additional hearing loss during the first 2 infusions. One patient had back pain occuring within the first infusion. Of the 15 patients with primary nonmetastatic OS and treated with the addition of mifamurtide to chemotherapy, 13 showed a complete remission, and 2 patients were still under treatment with a complete remission. Of 3 patients with progressive disease, 2 died while the disease progressed further in the third case over a 51-month period. The 3-year overall survival and event-free survival distributions were 87.5% (mean follow-up time, 46.12; 95% confidence interval, 37.79-52.45 mo) and 75.6% (mean follow-up time, 31.30; 95% confidence interval, 26.54-36.06 mo), respectively. We consider that mifamurtide therapy is a safe and well-tolerated agent in childhood OS.

Characterization and evaluation of amphotericin B loaded MDP conjugated poly(propylene imine) dendrimers.

This paper describes a novel strategy for targeted delivery of amphotericin B (AmB) to macrophages with muramyl dipeptide (MDP) conjugated multimeric poly(propyleneimine) (PPI) dendrimers. Synergistic antiparasitic activity due to immunostimulation by multimeric presentation of MDP on dendrimers was anticipated. MDP conjugated 5.0G PPI (MdPPI) dendrimers were synthesized and characterized. Therapeutic activity and toxicity of dendrimeric formulation of AmB (MdPPIA) were compared with marketed formulations of AmB. Highly significant (P<0.01) reduction in toxicity was observed in hemolytic toxicity and cytotoxicity studies in erythrocytes and J774A.1 macrophage cells, respectively. Formulation MdPPIA showed appreciable macrophage targeting potential and higher or equivalent antiparasitic activity against parasite infected macrophage cell lines and in vivo infection in Balb/c mice. These results suggest the developed MDP conjugated dendrimeric formulation of AmB as a promising immunostimulant targeted drug delivery system and a safer alternative to marketed formulations. From the clinical editor: Parasitic infections remain a significant issue in the clinical setting. The authors in this article studied the use of ligand anchored dendrimeric formulation of Amphotericin B to target infected macrophages and showed reduced toxicity, high anti-leishmanial activity. This may add another treatment option to available formulations in the future.

Pharmacokinetics and pharmacodynamics of liposomal mifamurtide in adult volunteers with mild or moderate hepatic impairment.

AIMS: To evaluate the pharmacokinetics and pharmacodynamics after a single dose of liposomal mifamurtide (liposomal muramyl tripeptide phospatidyl ethanolamine; MEPACT(®)) in adult subjects with mild (Child-Pugh Class A) or moderate (Child-Pugh Class B) hepatic impairment in comparison with age-, weight- and sex-matched healthy subjects with normal hepatic function. METHODS: Subjects received a 4 mg dose of liposomal mifamurtide via 1 h intravenous infusion. Blood samples were collected over 72 h for pharmacokinetic and pharmacodynamic assessments (changes in serum interleukin-6, tumour necrosis factor-α and C-reactive protein). RESULTS: Thirty-seven subjects were enrolled: nine with mild hepatic impairment, eight with moderate hepatic impairment and 20 matched healthy subjects. Geometric least-square mean ratios of total mifamurtide AUCinf for the mild hepatic impairment and moderate hepatic impairment groups vs. matched healthy subjects were 105% (90% confidence interval, 83.6-132%) and 119% (90% confidence interval, 94.1-151%), respectively, which are below the protocol-specified threshold (150%) to require development of dose-modification recommendations. Pharmacodynamic parameters for changes in serum interleukin-6 and tumour necrosis factor-α concentrations were generally similar across hepatic function groups. Mifamurtide-induced increases in serum C-reactive protein were attenuated in the moderate hepatic impairment group, consistent with the liver being the major organ of C-reactive protein synthesis. No grade ≥3 adverse events were seen in subjects administered mifamurtide (4 mg). CONCLUSIONS: These results support the conclusion that mild or moderate hepatic impairment does not produce clinically meaningful effects on the clinical pharmacokinetics or pharmacodynamics of mifamurtide; no dose modifications are needed in these special patient populations based on clinical pharmacological considerations.

A Protective Role of NOD2 on Oxazolone-induced Intestinal Inflammation Through IL-1ß-mediated Signalling Pathway.

BACKGROUND AND AIMS: NOD2 has emerged as a critical player in the induction of both Th1 and Th2 responses for potentiation and polarisation of antigen-dependent immunity. Loss-of-function mutations in the NOD2-encoding gene and deregulation of its downstream signalling pathway have been linked to Crohn's disease. Although it is well documented that NOD2 is capable of sensing bacterial muramyl dipeptide, it remains counter-intuitive to link development of overt intestinal inflammation to a loss of bacterial-induced inflammatory response. We hypothesised that a T helper bias could also contribute to an autoimmune-like colitis different from inflammation that is fully fledged by Th1 type cells. METHODS: An oedematous bowel wall with a mixed Th1/Th2 response was induced in mice by intrarectal instillation of the haptenating agent oxazolone. Survival and clinical scoring were evaluated. At several time points after instillation, colonic damage was assessed by macroscopic and microscopic observations. To evaluate the involvement of NOD2 in immunochemical phenomena, quantitative polymerase chain reaction [PCR] and flow cytometry analysis were performed. Bone marrow chimera experimentation allowed us to evaluate the role of haematopoietic/non-hematopoietic NOD2-expressing cells. RESULTS: Herein, we identified a key regulatory circuit whereby NOD2-mediated sensing of a muramyl dipeptide [MDP] by radio-resistant cells improves colitis with a mixed Th1/Th2 response that is induced by oxazolone. Genetic ablation of either Nod2 or Ripk2 precipitated oxazolone colitis that is predominantly linked to a lack of interferon-gamma. Bone marrow chimera experiments revealed that inactivation of Nod2 signalling in non-haematopoietic cells is causing a biased M1-M2 polarisation of macrophages and a decreased frequency of splenic regulatory T cells that correlates with an impaired activation of CD4 + T cells within mesenteric lymph nodes. Mechanistically, mice were protected from oxazolone-induced colitis upon administration of MDP in an interleukin-1- and interleukin-23-dependent manner. CONCLUSIONS: These findings indicate that Nod2 signalling may prevent pathological conversion of T helper cells for maintenance of tissue homeostasis.

A pharmacokinetic, pharmacodynamic, and electrocardiographic study of liposomal mifamurtide (L-MTP-PE) in healthy adult volunteers.

PURPOSE: This study evaluated the pharmacokinetics (PK), pharmacodynamics (PD), safety/tolerability, and cardiac safety of liposomal muramyl tripeptide phosphatidyl-ethanolamine [mifamurtide (L-MTP-PE)] in healthy adults. METHODS: L-MTP-PE 4 mg was administered intravenously over 30 min. Study participants were monitored from 24 h preinfusion until 72 h postinfusion. Blood samples were drawn over 0-72 h postdose to determine serum MTP-PE, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and C-reactive protein (CRP) concentrations. Electrocardiograpic (ECG) data were collected via continuous Holter monitoring beginning 24 h predose through 24 h postdose. Changes from time-matched pretreatment baseline QTc and associated two-sided 90 % confidence intervals were calculated. RESULTS: Twenty-one participants received L-MTP-PE. Total serum MTP-PE declined rapidly with a terminal half-life of 2.05 ± 0.40 h. PK variability was low, with <30 % coefficient of variation in systemic exposure. Serum concentrations of IL-6, TNF-α, and CRP increased following L-MTP-PE infusion. Maximum observed increases in IL-6 and TNF-α occurred at 4 and 2 h, respectively, returning toward baseline by 8 h postdose. L-MTP-PE was generally well tolerated, with no adverse events greater than grade 3. Headache, chills, tachycardia, nausea, and pyrexia were the most frequent adverse events. L-MTP-PE infusion resulted in an increased heart rate without readily apparent QTc prolongation. CONCLUSIONS: MTP-PE PK following L-MTP-PE administration were characterized by a short serum half-life and low variability. Increases in IL-6, TNF-α, and CRP and the safety profile were consistent with the immunomodulatory mechanism of action. No clinically significant effect of L-MTP-PE on cardiovascular repolarization was observed based on analysis of ECG QTc intervals.

[Use of the immunopotentiator N-acetyl-glucosamine-N-acetylmuramyl dipeptide during triple anti-Helicobacter pylori therapy].

AIM: To evaluate the efficiency of first-line Helicobacter pylori eradication therapy with glucosaminylmuramyldipeptide (Licopid JSC "Peptek", Russia). SUBJECTS AND METHODS: Eradication therapy was performed in 128 patients (84 men and 34 women; mean age 44.1 +/- 13.5 years) with duodenal bulb ulcer associated with H. pylori. The latter was detected in the gastroduodenal mucosa by a morphological study and rapid urease test before and 6-8 weeks after treatment and discontinuation of all drugs. Gastric metaplasia areas in the duodenum were revealed by periodic acid-Schiff and Alcian blue staining. The patients were divided into 4 groups according to the treatment protocol: 1) omeprazole (O) 0.04 g/day, clarithromycin (C) 1 g/day, amoxicillin (A) 2 g/day for 7 days (OCA7; n = 33); 2) the above drugs for 14 days (OCA14; n = 34); 3) O 0.04 g/day, C 1 g/day, A 2.0 g/day for 7 days, and glucosaminylmuramyldipeptide (Licopid) (L) 0.001 g/day for a day (OCA7L1; n = 34) and 4) the above drugs and L 0.01 g/day for 10 (OCA7L10; n = 27). RESULTS: According to the data of intention-to-treat analysis and per protocol, the H. pylori eradication rate was 81.8 and 87.1% for OCA7; 82.4 and 93.3% for OCA14; 88.2 and 93.8% for OCA7L1; 88.9 and 96% for OCA7L10 after PT and RRT, respectively. The rate of side effects was as follows: 6.1% for OCA7; 17.6% for OCA14 (5.9% stopped treatment); 5.9% for OCA7L1; 7.4% for OCA7L10. The cost of the treatment protocols was $ 32 for OCA7; $ 64 for OCA14; $ 40 for OCA7L1; $ 67 for OCA7L10. The intake of glucosaminylmuramyldipeptide (licopid) 0.001 g/day during 7-day triple anti-Helicobacter pylori therapy increased eradication by 6.4% (ITT) and 6.7% (PP), without raising the rate of side effects. CONCLUSION: H. pylori-positive patients with duodenal bulb ulcer should be given glucosaminylmuramyldipeptide (Licopid) 0.001 g/day during 7-day first-line eradication therapy as alternative to the 14-day treatment regimen.

Mifamurtide: osteosarcoma: ineffective and harmful.

The standard treatment for children and young adults with osteosarcoma consists of surgery, preceded and followed by methotrexate-based chemotherapy. Mifamurtide is an immunostimulant derived from a bacterial cell wall component. It is authorised in the European Union as an adjunct to combination chemotherapy after complete excision of non-metastatic osteosarcoma. Only one comparative, unblinded trial has been published, and its design was particularly complex. In a study population of 678 patients, adding mifamurtide to chemotherapy after tumour excision did not prolong the overall 6-year survival rate, which was about 75% with both treatments. Only serious adverse effects were collected, and they were not systematically recorded. Hypersensitivity reactions occurred in clinical trials, along with pleural and pericardial effusions, seizures, and muscle spasms. Severe hearing loss occurred in 12% of the patients treated with mifamurtide in the comparative trial, versus 7% of the other patients. In practice, given the lack of any survival benefit and the risk of serious adverse effects, it is better not to add mifamurtide to chemotherapy regimens used for treatment of osteosarcoma.

[Evaluation of the efficiency and reactogenicity of emulsion-based adjuvant systems in the manufacture of polyclonal enteroviral diagnostic sera].

Whether various adjuvants might be used in the manufacture of commercial enteroviral diagnostic sera (EDS) was studied. The following adjuvants: Ribi, SAF-1, and TiterMax were compared; vaseline-lanoline emulsion used to prepare EDSs, as well as modified Freund's complete adjuvant served as controls. Chinchilla rabbits were intramuscularly injected enterovirus antigens (enterovirus 70 and ECHO 2) together with the adjuvant emulsions. TiterMax showed the highest efficiency comparable with the activity of Freund's adjuvant. The activities of Ribi, SAF-1, and vaseline-lanoline emulsion were 3-4 times lower. The neutralizing activity of the sera obtained after 2-3 (TiterMax) or 4-5 (Ribi, SAF-1) immunizations was maximal. Further immunizations resulted in a reduction in the titers of neutralizing antibodies. TiterMax and vaseline-lanoline emulsion caused minimal complications at the site of inoculation whereas SAF-1 and Ribi gave rise to severer inflammatory responses.

Muramyl Tripeptide Plus Chemotherapy Reduces Metastasis in Non-Metastatic Osteosarcoma: A Single-Center Experience.

BACKGROUND: The immunomodulator mifamurtide plus a chemotherapy regimen has been shown to significantly improve the outcome in non-metastatic osteosarcoma patients. We report the results of the addition of mifamurtide to chemotherapy in newly diagnosed patients with osteosarcoma. METHODS: A total of 36 children with osteosarcoma without detectable metastasis were treated between November 2010 and April 2018 at the Ankara University Department of Pediatric Oncology. Mifamurtide was added to the chemotherapy regimen in 17 patients while the remaining 19 did not receive mifamurtide. The probabilities of metastasis and overall survival were compared between the groups. RESULTS: The 43-month survival rate was 87.5% and 89.9% in the patients who received and did not receive mifamurtide, respectively (p=0.65). Common side effects of mifamurtide were chills and fever. The addition of mifamurtide in the high-risk group with ≤95% necrosis tended to decrease the probability of distant metastasis (36.4% vs. 58.3%) (p=0.39). The time to metastasis in the group with positive surgical margins (4 months in one patient in the non-mifamurtide group, 7 and 20 months in the mifamurtide group) was also longer in the mifamurtide group. During the 43-month follow up period, median time to metastasis was longer in the mifamurtide group (20 vs. 5 months). In addition, mifamurtide plus chemotherapy decreased the risk of metastasis in the cases with primary site relapse. CONCLUSIONS: The addition of mifamurtide to chemotherapy might improve event-free survival by decreasing the probability of distant metastasis in bad histologic responders, and also by increasing the time to distant metastasis in the surgical margin positive group. Additional clinical studies are necessary to determine the long-term effects of mifamurtide on metastatic disease.
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Mifamurtide: CGP 19835, CGP 19835A, L-MTP-PE, liposomal MTP-PE, MLV 19835A, MTP-PE, muramyltripeptide phosphatidylethanolamine.

Mifamurtide is a conjugate of muramyl tripeptide linked to dipalmitoyl phosphatidyl ethanolamine; the phospholipid facilitates incorporation of the peptide into liposomes. The agent stimulates macrophages to seek out and destroy cancer cells. The compound was originated by Novartis (formerly CIBA-Geigy), and is being developed by IDM Pharma for osteosarcoma. Mifamurtide is being reviewed by regulatory authorities in the US and EU for this indication.CIBA-Geigy originally developed mifamurtide in the early 1980s and the agent was subsequently outlicensed to Jenner Biotherapies in the 1990s. IDM Pharma acquired the rights to the drug from Jenner in April 2003.IDM and Genesis Pharma have entered into an exclusive licensing and marketing agreement for mifamurtide in South East Europe. Under the agreement terms, IDM will receive an upfront fee from Genesis, as well as milestone payments on reaching certain sales levels in the territory. Medison Pharma signed an agreement with IDM Pharma for the sales and marketing of mifamurtide in Israel. IDM will receive an upfront license fee from Medison and will be entitled to receive a milestone payment upon regulatory approval of the agent in Israel, as well as royalties on net sales.IDM outlicensed exclusive marketing rights for mifamurtide in the UK and Ireland to Cambridge Laboratories in June 2005. In exchange, IDM is entitled to an upfront license fee and milestone payments prior to launch, as well as royalties calculated on product sales.Previously, Chiron Vaccines (a joint venture between Novartis and Chiron formed in 1995) investigated mifamurtide as an adjuvant in HIV gp120 vaccine; however, development has been discontinued.IDM Pharma will purchase approximately 7.1 million shares of its common stock to raise approximately $US23.5 million in net proceeds. The company intends to use the funds for working capital and corporate purposes, including the company's activities related to gaining marketing approval of mifamurtide in the US and Europe. Following the announcement by ODAC in May 2007, IDM Pharma decided to amend the NDA for mifamurtide with additional vital status data from the completed phase III trial. This data was not available at the time the original filing was made, and the company believes that capturing this supplemental data will overcome the need for additional trials, further confirm the overall survival benefit of mifamurtide in osteosarcoma, and provide evidence for approvability. IDM Pharma intends to analyse the additional follow-up data and submit an amendment to the agency by the first quarter of 2008; the company is also working on addressing other concerns raised by the US FDA in the non-approvable letter. The US regulatory submission included safety and efficacy data from NCI-funded phase III trials in 678 patients with osteosarcoma conducted by the Pediatric Oncology Group and the Children's Cancer Group in over 147 US centres. The NDA also included safety and biological effects data of mifamurtide from 17 phase I and II studies in 248 patients conducted by Ciba-Geigy. In the EU, IDM Pharma filed a MAA with the EMEA in November 2006 for approval of mifamurtide (Mepacttrade mark) in combination with postoperative chemotherapy for the treatment of patients with newly diagnosed osteosarcoma following complete surgical resection. The company expects that the EMEA will make a decision regarding marketing approval for mifamurtide by the end of 2007. Mifamurtide has orphan drug status for the treatment of osteosarcoma in the US and EU.

[Adverse immunologic effects of immunomodulators revealed in experiment and ways to their surmounting].

Immunomodulators licopid (synthetic analogue of muramylpeptide) and purified staphylococcal toxoid (PST) in some variants of experiments on mice caused adverse immunologic effects: enhancement of virus-induced immunosupression, shift from latent immunosupression (revealed only by low, but not standard, doses of test-antigen) to manifested one. Described adverse effects are not a contraindication for use of the studied drugs in practice. Their adverse effects were revealed only after single inoculation, whereas in clinical conditions PST and licopid are used by courses with duration of 5-7 and 10 days respectively. Our experiments show that inoculation of suppressive doses of the preparations repeated by 2-4 times can prevent the shift from latent to manifested immunosupression. Enhancement of immunosupression was not observed in case of combined administration of suppressive doses of PST and adjuvant dose of licopid.

Active specific immunotherapy of metastatic melanoma with an antiidiotype vaccine: a phase I/II trial of I-Mel-2 plus SAF-m.

PURPOSE: To determine the toxicity and immunologic activity of an antiidiotype melanoma vaccine that consists of monoclonal antibody I-Mel-2 (MELIMMUNE-2, IDEC Pharmaceuticals, La Jolla, CA) and an immunologic adjuvant SAF-m. PATIENTS AND METHODS: Twenty-six patients with metastatic melanoma, 17 of whom had previously received chemotherapy, were given 2 mg of I-Mel-2 and either 100 micrograms (n = 6) or 250 micrograms (n = 20) of SAF-m. Antiidiotype vaccine was given intramuscularly (IM) biweekly for 4 weeks, and then bimonthly until disease progression. Human antimurine antibodies (HAMA), anti-I-Mel-2 antibodies, and specific antibody (Ab)3 against the melanoma epitope mimicked by the vaccine were titrated before treatment, biweekly from weeks 4 to 12, and every 4 to 8 weeks thereafter. Computed tomographic (CT) scans of the chest, abdomen, and pelvis and magnetic resonance imaging (MRI) of the brain were obtained before and bimonthly during treatment to evaluate responses. RESULTS: Elevated titers of human antimouse antibodies and anti-I-Mel-2 antibodies were associated with clinical antitumor effect (P = .02 and P = .05, respectively). Ab3 was absent in most patients, but was found in the best clinical responder. Fever, myalgias/arthralgias, fatigue, nausea, and headaches were the most common toxicities. Grade III myalgias/arthralgias and headaches required dose reduction of SAF-m in eight patients at the 250-microgram dose. No treatment-related death occurred. Six patients had an antitumor effect: one complete response in liver and lung, two minor responses, and three stable disease. The patient with a complete response has survived nearly 5 years. CONCLUSION: I-Mel-2 antiidiotype vaccine was safe, tolerated best at the 100-microgram dose of SAF-m, and had immunologic and clinical activity.

Phase I trial of liposomal muramyl tripeptide phosphatidylethanolamine in cancer patients.

Twenty-eight evaluable patients with metastatic cancer refractory to standard therapy received escalating doses of muramyl tripeptide phosphatidylethanolamine (MTP-PE) (.05 to 12 mg/m2) in phosphatidylserine (PC):phosphatidylcholine (PS) liposomes (lipid:MTP-PE) ratio 250:1). Liposomal MTP-PE (L-MTP-PE) was infused over 1 hour twice weekly; doses were escalated within individual patients every 3 weeks as tolerated for a total treatment duration of 9 weeks. Routine clinical laboratory parameters, acute phase reactants and various immunologic tests were monitored at various time points during treatment. Toxicity was moderate (less than or equal to grade II) in 24 patients with chief side effects being chills (80% of patients), fever (70%), malaise (60%), and nausea (55%). In four patients L-MTP-PE treatment was deescalated due to severe malaise and recurrent fever higher than 38.8 degrees C. The maximum-tolerated dose (MTD) was 6 mg/m2. Significant (P less than .05) increases in WBC count, absolute granulocyte count, ceruloplasmin, beta 2-microglobulin, c-reactive protein, monocyte tumoricidal activity, and serum IL-1 beta were found. Significant decreases in serum cholesterol were also observed. Clearance of intravenously (iv)-infused technetium-99 (99mTc)-labeled liposomes containing MTP-PE in four patients was biphasic; gamma camera scans revealed uptake of radiolabel in liver, spleen, lung, nasopharynx, thyroid gland, and tumor (two patients). No objective tumor regression was seen. In view of its definite immunobiologic activity and lack of major toxicity, additional phase II and adjuvant trials of L-MTP-PE are warranted.

Effect of granulocyte-macrophage colony-stimulating factor inducer on left ventricular remodeling after acute myocardial infarction.

OBJECTIVES: We sought to determine the influence of granulocyte-macrophage colony-stimulating factor (GM-CSF) induction on post-myocardial infarction (MI) remodeling, especially in relation to the inflammatory response and myocardial fibrosis. BACKGROUND: Granulocyte-macrophage colony-stimulating factor modifies wound healing by promoting monocytopoiesis and infiltration of monocytes and macrophages into injured tissue; however, the effect of GM-CSF induction on the infarct healing process and myocardial fibrosis is unclear. METHODS: A model of MI was produced in Wistar rats by ligation of the left coronary artery. The MI animals were randomized to receive GM-CSF inducer (romurtide 200 microg/kg/day for 7 consecutive days) (MI/Ro) or saline (MI/C). RESULTS: Echocardiographic and hemodynamic studies on day 14 revealed increased left ventricular (LV) end-diastolic dimension, decreased fractional shortening, elevated LV end-diastolic pressure, and decreased LV maximum rate of isovolumic pressure development in MI/Ro compared with MI/C. Immunoblotting showed that expression of transforming growth factor (TGF)-beta1 in the infarcted site on day 3 after MI was decreased in MI/Ro compared with MI/C. In the infarcted site, TGF-beta1, collagen type I and type III messenger ribonucleic acid (mRNA) expression on day 3, and collagen content on day 7 were reduced in MI/Ro compared with MI/C, in association with marked infarct expansion. In MI/Ro, monocyte chemoattractant protein-1 mRNA level and the degree of infiltration of monocyte-derived macrophages (ED-1-positive)were greater in the infarcted site on day 7 than those in MI/C. CONCLUSIONS: The GM-CSF induction by romurtide facilitated infarct expansion in association with the promotion of monocyte recruitment and inappropriate collagen synthesis in the infarcted region during the early phase of MI.

A novel immunostimulator, N-[alpha-O-benzyl-N-(acetylmuramyl)-L-alanyl-D-isoglutaminyl]-N6-trans-(m-nitrocinnamoyl)-L-lysine, and its adjuvancy on the hepatitis B surface antigen.

N(2)-[alpha-O-benzyl-N-(acetylmuramyl)-L-alanyl-D-isoglutaminyl]-N(6)-trans-(m-nitrocinnamoyl)-L-lysine (muramyl dipeptide C, or MDP-C) has been synthesized as a novel, nonspecific immunomodulator. The present study shows that MDP-C induces strong cytolytic activity by macrophages on P388 leukemia cells and cytotoxic activity by cytotoxic T lymphocytes (CTLs) on P815 mastocytoma cells. Our results also indicate that MDP-C is an effective stimulator for production of interleukin-2 and interleukin-12 by murine bone marrow derived dendritic cells (BMDCs) and production of interferon-gamma by CTLs. Additionally, MDP-C increases the expression levels of several surface molecules, including CD11c, MHC class I, and intercellular adhesion molecule-1 in BMDCs. Moreover, MDP-C remarkably enhances the immune system's responsiveness to hepatitis B surface antigen (HBsAg) in hepatitis B virus transgenic mice for both antibody production and specific HBsAg T-cell responses ex vivo. Our results indicate that MDP-C is an apyrogenic, nonallergenic, and low-toxicity immunostimulator with great potential for diagnostic, immunotherapeutic, and prophylactic applications in diseases such as hepatitis B and cancers.

Phase I trial of ImmTher, a new liposome-incorporated lipophilic disaccharide tripeptide.

A phase I clinical trial was conducted to evaluate the toxicology and biological activity of a new liposome-incorporated lipophilic disaccharide tripeptide, ImmTher. Twelve patients with advanced nonhematological malignant disease received 13 courses of therapy at dose levels of 200-1,200 micrograms/m2. A course of therapy consisted of once-weekly administration of the drug for 2-12 weeks. The major clinical toxicities observed were chills and hypotension. No renal, hepatic, cardiac, or hematological toxicity was observed. A small decrease in pulmonary diffusion capacity was observed. Biological activity was demonstrated by changes in plasma cytokine levels, changes in in vitro monocyte cytotoxicity, and by a decrease in tumor size. Improvement was observed in three of three patients with metastatic disease to the liver. Response in these three patients correlated with an increase in their tumor necrosis factor and neopterin levels compared with nonresponders. These preliminary indications of biological and clinical activity of a liposome-incorporated lipophilic disaccharide tripeptide in patients with advanced metastatic hepatic disease suggest a potential new therapeutic approach to this common problem.

Clinical tolerance and profile of cytokine induction in healthy volunteers following the simultaneous administration of ifn-alpha and the synthetic immunomodulator murabutide.

As the therapeutic use of interferon-alpha (IFN-alpha) is limited by a dose-dependent toxicity and variable efficacy, ways of improving the therapeutic index of the cytokine are being sought. Murabutide (N-acetyl muramyl-L-alanyl-D-glutamine-O-n-butyl-ester) (ISTAC Biotechnology, Lille, France) is a safe synthetic and clinically acceptable immunomodulator that enhances the biologic activities of IFN-alpha in different experimental models. We evaluated in healthy human volunteers tolerance of the coadministration of Murabutide with increasing doses of IFN-alpha. The simultaneous administration of the two drugs was well tolerated without any increased or prohibiting toxicity, and all recipients experienced side effects that were similar to those observed after the administration of IFN-alpha alone. We also profiled the serum levels of cytokines induced following coinjection of the two drugs. We mostly detected an induction of anti-inflammatory cytokines and of human immunodeficiency virus type 1 (HIV-1)-suppressive beta-chemokines, in the absence of release of key proinflammatory cytokines. Therefore, the simultaneous administration of Murabutide and IFN-alpha is well tolerated and does not lead to increased toxicity. In addition, the selectivity in the profile of cytokines and chemokines induced following the coadministration of Murabutide and IFN-alpha points to the potential use of this combination in the treatment of inflammatory diseases and chronic viral infections.

Phase II study of liposomal muramyl tripeptide phosphatidylethanolamine (MTP/PE) in advanced soft tissue sarcomas of the adult. An EORTC Soft Tissue and Bone Sarcoma Group study.

The EORTC Soft Tissue and Bone Sarcoma Group conducted a phase II study with intravenous muramyl tripeptide phosphatidylethanolamine (MTP/PE) at a dose of 4 mg once weekly in 20 patients with metastatic soft tissue sarcomas. Responses were not seen in 19 evaluable patients. Toxicity consisted mainly of a mild flu-like syndrome after 62% of drug administrations. It is concluded that MTP/PE at this dose and schedule has no activity in metastatic soft tissue sarcoma.