Mannide Monooleate Research Articles

Tridecylcyclohexane in incomplete Freund's adjuvant is a critical component in inducing experimental autoimmune diseases.

Incomplete Freund's adjuvant (IFA) has been used for many years to induce autoimmune diseases in animal models, including experimental autoimmune encephalitis and collagen-induced arthritis. However, it remains unclear why it is necessary to emulsify autoantigen and heat-killed Mycobacterium tuberculosis (HKMtb) with IFA to induce experimental autoimmune diseases. Here, we found that immunization with self-antigen and HKMtb was insufficient to induce autoimmune diseases in mice. Furthermore, IFA or one of its components, mineral oil, but not mannide monooleate, was required for the development of experimental autoimmune disease. Immunization with autoantigen and HKMtb emulsified in mineral oil facilitated innate immune activation and promoted the differentiation of pathogenic CD4+ T cells, followed by their accumulation in neuronal tissues. Several water-soluble hydrocarbon compounds were identified in mineral oil. Of these, immunization with HKMtb and autoantigen emulsified with the same amount of hexadecane or tridecylcyclohexane as mineral oil induced the development of experimental autoimmune encephalitis. In contrast, immunization with HKMtb and autoantigen emulsified with tridecylcyclohexane, but not hexadecane, at doses equivalent to those found in mineral oil, resulted in neuronal dysfunction. These data indicate that tridecylcyclohexane in mineral oil is a critical component in the induction of experimental autoimmune disease.

Cellular uptake of self-assembled phytantriol-based hexosomes is independent of major endocytic machineries

Despite increasing interests in non-lamellar liquid crystalline dispersions, such as hexosomes, for drug delivery, little is known about their interactions with cells and mechanism of cell entry. Here we examine the cellular uptake of hexosomes based on phytantriol and mannide monooleate by HeLa cells using live cell microscopy in comparison to conventional liposomes. To investigate the importance of specific endocytosis pathways upon particle internalization, we silenced regulatory proteins of major endocytosis pathways using short interfering RNA. While endocytosis plays a significant role in liposome internalization, hexosomes are not taken up via endocytosis but through a mechanism that is dependent on cell membrane tension. Biophysical studies using biomembrane models highlighted that hexosomes have a high affinity for membranes and an ability to disrupt lipid layers. Our data suggest that direct biomechanical interactions of hexosomes with membrane lipids play a crucial role and that the unique morphology of hexosomes is vital for their membrane activity. Based on these results, we propose a mechanism, where hexosomes destabilize the bilayer, allowing them to “phase through” the membrane. Understanding parameters that influence the uptake of hexosomes is critical to establish them as carrier systems that can potentially deliver therapeutics efficiently to intracellular sites of action.

Characterization of Lipid-Based Hexosomes as Versatile Vaccine Carriers

Subunit vaccines typically show insufficient immunogenicity. To address this issue, we developed a novel self-adjuvanting particulate carrier system based upon the lipids phytantriol (Phy) and mannide monooleate (MaMo). Phy is a lipid known to form nonlamellar phases in fully hydrated systems, whereas MaMo has been found to promote immune responses in emulsion form. A bulk phase composition of Phy/MaMo (14 wt %) showed hexagonal (HII) phase behavior over a practical temperature range (including room and body temperature), and was therefore used for particle development. Hexosomes stabilized with different concentrations of either poloxamer 407, Myrj 59, or Pluronic F108 were successfully prepared. To demonstrate the versatile nature of these systems, the particles were further modified with either positively or negatively charged lipids and loaded with model antigens, while maintaining the HII structure. These hexosomes are structurally robust and amenable to customization, rendering them suitable as antigen delivery carriers.

Age-Dependent Changes in 24-Hour Rhythms of Thymic and Circulating Growth Hormone and Adrenocorticotropin in Rats Injected with Freund’s Adjuvant

Objective: To analyze the 24-hour changes in thymic and serum concentration of growth hormone (GH) and adrenocorticotropin (ACTH) and their correlation with thymic concentrations of glutamate, aspartate, taurine and GABA in young and old rats during the acute phase of adjuvant’s arthritis. Methods: Young (50-day-old) and old (18-month-old) rats were injected subcutaneously with Freund’s adjuvant or its vehicle (paraffin oil containing 15% mannide monooleate). Eighteen days later, they were killed at six different time intervals throughout a 24-hour cycle. Serum and thymic levels of GH and ACTH were measured by radioimmunoassay. Thymic amino acid concentration was measured by HPLC. A quantitative assessment of arthritis was made in an independent group of rats by plethysmography. Results: Old rats injected with Freund’s adjuvant exhibited fewer clinical signs of inflammation than young rats. Significant 24-hour changes in thymic and serum GH occurred, except for serum GH in adjuvant’s vehicle-treated old rats. Aging augmented thymic GH and decreased serum GH. Immunization with Freund’s adjuvant did not modify GH concentration. Thymic and serum concentration of GH correlated negatively. Thymic ACTH varied significantly over 24 h with maxima during the dark phase, except in Freund’s adjuvant-treated young rats. Maximal serum ACTH levels occurred in the late afternoon except in Freund’s adjuvant-treated old rats which showed maxima at night. Immunization with Freund’s adjuvant augmented thymic and circulating concentrations of ACTH. Thymic and serum concentration of ACTH correlated positively. Thymic concentration of glutamate, aspartate and taurine decreased in aged rats and correlated significantly with thymic ACTH. Conclusion: The results support the existence of a thymic compartment of GH and ACTH that may be independently regulated.

Preformulation Studies on Freund's Incomplete Adjuvant Emulsion

Freund's Incomplete Adjuvant (FIA), which is used in vaccine therapy, is a water-in-oil emulsion delivery system consisting of an aqueous internal phase containing an antigenic protein dispersed in an external phase containing a mixture of mannide monooleate and light mineral oil. Preformulation studies are reported in this investigation for FIA emulsion. The preformulation studies included the determination of the critical micelle concentration (CMC) of the formulations investigated, the surface activity of mannide monooleate at the interface between the oil phase and the aqueous phase containing ovalbumin as the model antigenic protein, and the effect of ovalbumin on the surface activity at the interface. The influence of the concentration of mannide monooleate and/or ovalbumin on the interfacial tension between light mineral oil and either purified water or 0.9% w/v normal saline solution was measured by the DuNouy Ring Method at 25°C. The CMC was determined experimentally from the relationship between the concentration of the surface active agent in each formulation and the interfacial tension. The number of moles of the surface active agent per unit area at the interface (surface excess concentration) was calculated from the Gibbs' Adsorption equation. The results indicated that mannide monooleate was an effective surface active agent since the formulation containing only mannide monooleate provided the lowest magnitude of CMC. The presence of the surface active agent, mannide monooleate and/or ovalbumin, in the formulations studied reduced the interfacial tension between the two phases. The surface activity was influenced by the presence of an electrolyte (sodium chloride), a protein (ovalbumin), or mannide monooleate in the formulation. The presence of antigenic proteins in the aqueous phase of a water-in-oil emulsion influenced the effectiveness of a surface active agent in the formulation.

Characterization of Mannide Monooleate

Two lots of a specially treated mannide monooleate emulsifier were fractionated by column chromatography and characterized by TLC and GC; individual components were identified by mass spectrometry. Silica gel fractionation indicated the presence of three major classes of components: diesters, monoesters, and polymeric carbohydrate material. The diester contents of Lots 114B and 115B were 36.3 and 51.8 wt. % while the monoester content was 52.2 and 31.4 wt. %, respectively. The carbohydrate polymer content amounted to 11.2 and 15.8 wt. %, respectively. The mannide monooleate (as the chemical entity) content within Lots 114B and 115B was 33.6 and 20 wt. %, respectively, while the mannide dioleate content varied from 21.9 wt. % (114B) to 29.2 wt. % (115B). Six cyclic fatty acids were observed in these two lots as well as in two toxic lots identified as 8B and 3589. The UV absorptions at 267, 278, 298, and 315 nm. were approximately fivefold greater for Lot 8B than Lot 114B and approximately 20‐fold greater for Lot 3589. Nonconjugated trans‐olefins were present in Lots 114B and 115B to the extent of 5.9 and 6.0 wt. %, respectively, while the conjugated tratts‐trans‐diene content amounted to about 1.6 wt. % for both specially treated lots. In both lots, 14 components were found to comprise the monoester class, while 13 components comprised the diester class. The carbohydrate material was not characterized.

Murine Thyroiditis: Importance of Adjuvant and Mouse Strain for the Induction of Thyroid Lesions

Abstract For the production of experimental autoimmune thyroiditis in the mouse comparison of two preparations of complete Freund's adjuvant showed that one, using n-hexadecane and glycerol mono-oleate with freshly harvested tubercle bacilli, was more effective in eliciting both dermal hypersensitivity and thyroid lesions than a mixture of paraffin oil and mannide mono-oleate with dried tubercle bacilli. Both preparations were of comparable efficiency in eliciting thyroid-specific antibodies detected by passive hemagglutination. With either adjuvant, randomly bred CF-1 mice were consistently more susceptible to the induction of thyroid lesions than were inbred C3H mice. When the paraffin oil adjuvant was used, C3H mice developed no lesions despite high titers of circulating antibody.

Metabolic Fate of Mineral Oil Adjuvants Using 14C-Labeled Tracers II: Mannide Monooleate

This investigation was undertaken to determine the metabolic fate of mannide monooleate when employed in a mineral oil emulsion. Female white rats and female squirrel monkeys were injected subcutaneously or intramuscularly with an emulsion made with mineral oil and surfactant and including either 1-14C-oleate or UL-14C-mannide-labeled mannide monooleate tracer preparations. It was shown that 30-40% of the surfactant mixture is removed from the site of injection after 24 hr. After 1 week, 40-60% of the surfactant is removed from the site of injection; while after 3 months, 10-30% of the surfactant still remains. The 1-14C-oleatelabeled mannide monooleate was largely incorporated into the various lipid classes, while the UL-14C-mannide-labeled mannide monooleate preparation was largely eliminated in the urine. There was some indication that the inguinal lymph nodes of monkeys may have contained unusually large amounts of radioactivity.

Metabolic Fate of Mineral Oil Adjuvants Using 14C-Labeled Tracers I: Mineral Oil

This investigation was undertaken to determine the metabolic fate of injected mineral oil using n-hexadecane as the tracer. Female white rats and female squirrel monkeys were injected either subcutaneously or intramuscularly with an emulsion containing 1-14C-n-hexadecane made with mineral oil and mannide monooleate. The amount of radioactivity remaining at the site of injection and the distribution of radioactivity in the major organs were determined at varying time periods up to 10 months after injection. One week after injection, 85-98% of the 1-14C-n-hexadecane-labeled mineral oil remained at the site of injection. After 3 months, 50-60% of the mineral oil remained; and after 10 months, approximately 25-30% of the labeled mineral oil was still at the site of injection. The mineral oil tracer, which migrated from the site of injection, was readily incorporated into lipids. Aside from the site of injection, there did not appear to be any accumulation of the 1-14C-n-hexadecane-labeled mineral oil in the major organs.

Influence of antigens on release of free fatty acids from Arlacel A (mannide monooleate).

SummaryWith use of the modified method of Dumcombe for assay of FFA, it was found that water-in-oil emulsified tetanus toxoids, cholera and typhoid vaccines which induced abscesses in human beings contained more FFA than did emulsified products not inducing abscesses. The purer the parent tetanus toxoid, the lower was the free fatty acid content of the emulsified antigen. C. tetani culture filtrates, cholera vaccine, one typhoid vaccine and several types of allergenic extracts promoted the release of FFA from the emulsifier, Arlacel A. Preliminary data suggest that the hydrolytic component of C. tetani filtrates is an enzyme. The relationship of FFA released by the interaction of antigens on the emulsifying agent of the oil adjuvant to untoward reactivity remains to be determined.

Water/Oil Emulsions Prepared by Low Pressure Capillary Homogenization II: Stabilizing Influence of Inorganic Electrolytes, Secondary Emulsifiers, and Temperature

Aqueous dispersions in light mineral oil stabilized with mannide mono-oleate were prepared under a set of standard conditions and evaluated by rotational viscometry in conjunction with optical microscopy. lnorganic electrolytes in the aqueous internal phase, at concentrations as low as 0.01 M, increased apparent viscosity, retarded sedimentation, and had a marked stabilizing influence. The addition of small amounts of water-soluble surfactants to the internal phase yielded extremely fine dispersions, but these agents decreased stability and tended to cause inversion. Storage of w/o emulsions at 5° had a definite stabilizing influence when compared to room temperature storage. Aqueous dispersions in light mineral oil stabilized with mannide mono-oleate were prepared under a set of standard conditions and evaluated by rotational viscometry in conjunction with optical microscopy. lnorganic electrolytes in the aqueous internal phase, at concentrations as low as 0.01 M, increased apparent viscosity, retarded sedimentation, and had a marked stabilizing influence. The addition of small amounts of water-soluble surfactants to the internal phase yielded extremely fine dispersions, but these agents decreased stability and tended to cause inversion. Storage of w/o emulsions at 5° had a definite stabilizing influence when compared to room temperature storage.

Water/Oil Emulsions Prepared by Low Pressure Capillary Homogenization I: Effects of Emulsator and Composition Variables on Mannide Mono-Oleate Stabilized Systems

An automated reciprocating capillary emulsator is described which duplicates the low-pressure homogenization principle of the interconnected glass syringe method used to prepare repository w/o emulsions. The w/o emulsions produced by this emulsator were evaluated by rotational viscometry and optical microscopy. Rheological flow curves of concentrated emulsions could be correlated with the extent of dispersion and degree of coalescence. Coalescence rates were found to be an inverse function of the surfactant concentration, with 20 per cent mannide mono-oleate furnishing the best relative stability for water-in-mineral oil emulsions. The rate of shear was found to govern the degree of emulsification, with a short capillary of large diameter producing the finest dispersion.

NEW METABOLIZABLE IMMUNOLOGIC ADJUVANT FOR HUMAN USE. I. DEVELOPMENT AND ANIMAL IMMUNE RESPONSE.

SummaryA new immunologic adjuvant, adjuvant 65, is described and the results of extensive tests of polyvalent influenza virus vaccine in the adjuvant are presented. The vaccine consists of a water-in-oil emulsion in which the adjuvant portion consists of peanut oil, mannide monooleate and aluminum monostearate. The adjuvant effect measured in terms of hemagglutination-inhibition antibody responses 28 days after vaccination in mice or guinea pigs is roughly equal to that obtained with Freund's incomplete mineral oil adjuvant. Adjuvant 65 influenza vaccine showed striking enhancement and long-term persistence of antibody in guinea pigs, rabbits and monkeys compared with the corresponding aqueous vaccines. The findings in histomorphologic-pathologic studies in animals and the clinical and serologic results obtained in large-scale studies in man are presented elsewhere. Adjuvant 65 shows promise of meeting the present urgent need for a safe and effective adjuvant for enhancing immunologic responses to viral a...

Antibody Formation and Sensitization with the Aid of Adjuvants

Summary and Conclusions Antibody formation was enhanced and sustained when the antigen (typhoid bacillus) was incorporated into a water-in-oil emulsion prepared with paraffin oil and Falba or anhydrous lanolin or Arlacel A (mannide monooleate) or myricin combined with cholesterol. Oil-in-water emulsion prepared with Tween 80 (polyoxyethylene sorbitan monooleate) and paraffin oil was ineffective. When oleic acid combined with myricin was used as an emulsifying agent antibody formation was neither enhanced nor sustained. Emulsions prepared with “lecithin” and paraffin oil are unstable mixed water-in-oil and oil-in-water emulsions. Antibody formation with such emulsions was highly accelerated but not as well sustained as with Falba and paraffin oil. Antibody formation is scant when peanut oil is substituted for paraffin oil. The addition of killed mycobacteria, namely M. butyricum or M. tuberculosis or M. phlei to water-in-oil emulsions further augments antibody formation. Lipid extract from M. tuberculosis has a similar augmenting effect. Cholesterol also increases the adjuvant effect of water-in-oil emulsion. A large amount of cerotic acid added to water-in-oil emulsions retards antibody formation. In experiments with a small number of guinea pigs sensitization to picryl chloride was produced only by emulsion containing killed tubercle bacilli. Other substances which augment antibody formation such as lipid extract from M. tuberculosis and cholesterol did not produce this effect. The enhancing effect on antibody formation and the alteration of sensitization may occur concomitantly but they appear to be distinct phenomena.

Fate of Mannide Monooleate in the Animal Body

SummaryIn the quantities administered in these experiments mannide monoiileate appears to be neither acutely nor chronically toxic to the white rat and to be innocuous in the diet of the Rhesus macacus monkey. Mannide monoijleate is absorbed from the intestinal tract of the white rat. No damage was observed to the important viscera of the white rat or monkey after an 8-week feeding period with mannide monoijleate.