Monophosphoryl lipid A

Hamdy S, Elamanchili P, Alshamsan A et al (2007) Enhanced antigen-specific primary CD4+ and CD8+ responses by codelivery of ovalbumin and toll-like receptor ligand monophosphoryl lipid A in poly(D, L-lactic-co-glycolic acid) nanoparticles. J Biomed Mater Res A 81 652-662... 

A disadvantage of the derivatization procedure used in the case of S. typhimurium (60) was that the trimethylsilylated dimethyl monophosphoryl lipid A derivative was not suitable for the characterization of the (free) hydroxyl group in position 4. This identification could be achieved, however, by the approach of Imoto et al. (35), who investigated the dimethyl monophosphoryl lipid A of E. coli and its per-O-acetylated derivative to compare the chemical shifts before and after acetylation. From the differences in the chemical shift observed in H-n.m.r., the authors could clearly demonstrate that, in their preparation, position 4 of GlcN(I) was free. All... 

Friede M, et al. Induction of immune response against a short synthetic peptide antigen coupled to small neutral liposomes containing monophosphoryl lipid A. Mol Immunol 1993 30 539. 

The influence of physicochemical properties of liposomes, such as charge density, membrane fluidity, and epitope density, on the immune response elicited by antigens has been extensively studied [37]. In addition to antigens, other immune stimulators that are amphoteric muramyl peptides or lipid-soluble compounds, such as monophosphoryl lipid A or muramyl tripeptidyl-phosphatidylethanolamine, can also be incorporated into liposomes to increase their adjuvant effect in eliciting immune responses [34]. 

Therapeutic vaccines were tested in BALB/c mice bearing TA3-Ha mammary carcinoma. The treatment consisted of 4 subcutaneous injections, at 3-6 days intervals, of Detox [a commercial preparation of cell wall skeletons from Mycobacterium phlei and non-toxic monophosphoryl lipid A from Salmonella minnesota (S. minnesota) in squalane oil and Tween 80 from Ribi Immunochemical research, Montana, USA] mixed with Thomsen-Friedenreich (TF) antigen coupled with KLH (Keyhole Limpet Hemocyanin) performed 5 days after the tumor cell injection. This vaccination achieved the survival of 25 % of the mice. Pretreatment of mice with cyclophosphamide in order to inhibit any suppressive response, increased survival to 50 % when the treatment began 5 days after tumor cell injection, and to 90 % when the treatment began 2 days after tumor cell injection. Both antibody as well as delayed-... 

In C3H/HeN mice bearing MH134 hepatoma, monophosphoryl lipids A from P. gingivalis or S. minnesota Re 595 increased the survival of mice when administered in combination with tumor cell lysates and Freund s incomplete adjuvant [31],... 

A phase I trial was conducted with monophosphoryl lipid A (MPLA) prepared from S. typhimurium or S. minnesota injected i.v. in patients with disseminated cancer. Fever, chills and fatigue were Ihe most common side effects and the dose of 250 pgfai2 i..e. (250x1.7) 65=6 pglcg was estimated acceptable [189]. 

Trials of therapeutic vaccination against prostate cancer used OncoVax-P (Jenner Biotherapies, Inc, San Ramon, California). OncoVax-P consists of 200 pg monophosphoryl lipid A (similar to that used in Detox) added to 1 ml liposomes and 100 pg PSA (prostate-specific antigen). Patients received injections by different routes (intramuscular, intravenous or subcutaneous) according to the trial, with or without GM-CSF, IL-2 or BCG and cyclophosphamide pretreatment. No serious side effects were seen. DTH and antibody responses were achieved. Vaccination increased the PSA-reactive T cell frequency as determined by IFN-y secretion, but no toxicity against PSA-expressing target cells was detected. The most effective strategy could not be determined, and no conclusion about the clinical efficacy of the treatment was possible [214,215],... 

Immunostimulatory adjuvants exert their effects predominantly at the cytokine level or through the activation of costimulatory signals. The type of response required for optimal protection depends on the pathogen. One class of immunostimulatory adjuvants is derived from the lipopolysaccharide of gram-negative bacteria. The most extensively evaluated member of this family, monophosphoryl lipid A (MPL), is obtained from Salmonella minnesota. MPL has been shown to induce the synthesis and release of cytokines, which promote the generation of specific immune responses. 

Abbreviations. MPL, monophosphoryl lipid A PLG, poly(lactide-co-glycolide) CpG ODN, CpG oligo-deoxynucleotide. 

Ulrich JT, Myers KR. Monophosphoryl lipid A as an adjuvant past experiences and new directions. Pharm Biotechnol 1995 6 495-524. 

Figure 1 shows the scheme for the preparation of purified lipid A from endotoxin. S. typhimurium G30/C21 was extracted by the method of Galanos t aK (24) and submitted to one of two different conditions of hydrolysis (a) 0.1 N HC1 [in methanol-water (1 1, v/v)], 100 °C, 45 min, to yield the crude monophosphoryl lipid A (nontoxic), and (b) 0.02 M sodium acetate, pH 4.5, 100 °C for 30 min (two cycles) to yield the crude diphosphoryl lipid A (toxic). The 0.1 N HC1 hydrolysis product was fractionated on a Sephadex LH-20 column (23). Each of these fractions was then separated by preparative thin layer chromatography (TLC) on silica gel H (500 ym), with the solvent system chloroform-methanol-waterconcentrated ammonium hydroxide (50 25 4 2, v/v) as previously described (23) to yield TLC fractions 1-7 and 1-9 respectively. 

The results of the chemical analysis of these fractions are given in Table VII. For the monophosphoryl lipid A (TLC-1, -3, and -5), the glucosamine phosphate ratio was 1.98 to 2.15, whereas... 

Figure 2. Reverse-phase HPLC of purified [l4C]monophosphoryl lipid A. A, TLC-3 B, TLC-5. The sample sizes were 1.4 and 2 mg, respectively (60,000 cpm each). The labeled lipid As were obtained from S. typhimurium G30/G21 grow n in the presence of[l-,4C]acetate as previously described (23). 

High Performance Liquid Chromatography (HPLC) of Monophosphoryl Lipid A... 

The analysis of the purified monophosphoryl lipid A by reverse-phase HPLC revealed the degree of purity of the fractions TLC-1, -3, -5, and -7 (23). TLC-1 and -3 each gave one major peak (85%), and these peaks had identical elution times. TLC-5 showed one major (72%) and one minor (18%) peak. TLC-7 resolved into one major (48%) and two minor (21 and 16%) peaks. One of the minor peaks in TLC-7 (16%) was the overlapping contaminant of the minor components in TLC-5. Representative results of the HPLC analysis of C-labeled TLC-3 and -5 are shown in Figure 2. 

The TLC purified diphosphoryl lipid A was hydrolyzed in 0.1 N HCl at 100 °C for 30 min to yield the corresponding monophosphoryl lipid A derivatives as previously described (23). These products were then compared with previously characterized monophosphoryl lipid A fractions by TLC using silica gel H (250 Vim) and the previously mentioned solvent system. The TLC fractions -3, -5, -7 of the acid hydrolyzed diphosphoryl lipid A series corresponded with a similarly numbered series of monophosphoryl lipid A fractions (TLC-3, -5, and -7). There appeared to be some breakdown of the monophosphoryl lipid A products to the lower homologues presumably by the acid catalyzed hydrolysis of some fatty ester linkages. TLC-1 and -9 were not analyzed due to small sample sizes. 

The TLC purified diphosphoryl lipid A fractions (TLC -3, -5, and -7) were analyzed by FAB mass spectrometry in the negative mode as previously described (23) and the results are shown in Table X. TLC-3 gave a molecular ion (M-H) at m/z 1796 TLC-5, m/z 1586 TLC-7, m/z 1360. As expected, these values were 80 amu or (P03H2 H) larger than those for the corresponding monophosphoryl lipid A s of the series as shown in Table VIII. These results established the structural relationship between the mono- and diphosphoryl lipid A s. 

The results of the biological tests carried out on the purified monophosphoryl lipid A fractions are shown in Table XI. The chick embryo lethality test showed that both TLC-1 and -3 were nontoxic, whereas TLC-5 exhibited some toxicity. These results indicate that there might be two levels of toxicity based on structure. The presence or absence of the sugar 1-phosphate group (and possibly some other unknown group) would control the upper... 

Figure 3. Structure of nontoxic monophosphoryl lipid A. R, = 3-hydroxymyristoyl R2 = lauroyl, 3-myristoxymyristoyl, or H.

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