Solvent-Assisted Hot Melt Extrusion of a Thermally Labile, High Melting Point Compound.

Molecular dispersions are a highly effective method of increasing bioavailability for a poorly soluble active pharmaceutical ingredient (API) and can be prepared on a large scale by hot melt extrusion (HME). Processing thermally labile active pharmaceutical ingredients (APIs) via HME is generally more difficult, with operating temperatures limited to below that of the API melting point. API melting is considered essential to facilitate the formation of a fully homogeneous amorphous system. Processing below the melting point renders the system much more susceptible to residual crystalline content; hence, HME is not suitable for APIs which degrade upon melting. In the following work, meloxicam (MEL) was used as a model API, possessing properties of high melting temperature and thermal lability. In this proof of concept work, a modified HME method, termed solvent-assisted HME, was used to overcome this issue and prepare an amorphous solid dispersion using HME, wherein a solvent was incorporated in the formulation blend during extrusion and removed post-processing. Formulations containing 10%wt meloxicam (MEL) and 90%wt polyvinylpyrrolidone vinyl acetate (PVPVA) copolymer were extruded using a twin-screw extruder at temperatures below the melting point of MEL. Dimethylformamide (DMF) solvent was added directly into the extruder barrel through a liquid addition port, resulting in extrudate products having a higher conversion of API to the amorphous form. The incorporation of solvent allowed a significant reduction in processing temperatures due to its increased mobility, while also driving the conversion of the API to its amorphous form. The solvent was successfully reduced through a secondary drying step using a vacuum oven. This advancement has demonstrated the potential for thermally labile APIs to be processed via HME expanding the applications of this technology.

Fixed Dose Combination Formulations: Multilayered Platforms Designed for the Management of Cardiovascular Disease.

Hyperlipidaemia is considered as one of the main risk factors associated with cardiovascular diseases (CVDs). Among different lipid-lowering agents used to manage hyperlipidaemia, statins are highly prescribed for management of hyperlipidaemia with simvastatin being one of the most common. Simvastatin is susceptible to extensive metabolism by CYP450 3A4 and 3A5, which are expressed both in the liver and the gastrointestinal tract. Nevertheless, the localization of these enzymes is site-dependent with lower concentration at the distal/proximal regions of the small intestine/colon. In addition to statins, medications such as antihypertensive agents and anticoagulants are introduced as adjuvants, for the treatment of cardiovascular disease. The aim of this study was to design a bilayer delivery system capable of delivering biphasic release of simvastatin and aspirin, within a fixed dose combination. A delayed release platform based on a combination of anionic polymers prepared using hot-melt extrusion was developed to delay the release of simvastatin. An optimized formulation tested for dissolution performance clearly demonstrated an ability to delay the release of simvastatin. In addition, an immediate release layer based on Kollidon VA64 was successfully developed to deliver aspirin. Both formulations were then manufactured as a bilayer drug delivery system (tablets and coextrudates), and the release performance was examined. On the basis of the obtained results, these formulations may be used as a platform for delivering a wide range of medications in a biphasic manner.

Gastroretentive extended-release floating granules prepared using a novel fluidized hot melt granulation (FHMG) technique.

The objective of this work was to investigate the feasibility of using a novel granulation technique, namely, fluidized hot melt granulation (FHMG), to prepare gastroretentive extended-release floating granules. In this study we have utilized FHMG, a solvent free process in which granulation is achieved with the aid of low melting point materials, using Compritol 888 ATO and Gelucire 50/13 as meltable binders, in place of conventional liquid binders. The physicochemical properties, morphology, floating properties, and drug release of the manufactured granules were investigated. Granules prepared by this method were spherical in shape and showed good flowability. The floating granules exhibited sustained release exceeding 10 h. Granule buoyancy (floating time and strength) and drug release properties were significantly influenced by formulation variables such as excipient type and concentration, and the physical characteristics (particle size, hydrophilicity) of the excipients. Drug release rate was increased by increasing the concentration of hydroxypropyl cellulose (HPC) and Gelucire 50/13, or by decreasing the particle size of HPC. Floating strength was improved through the incorporation of sodium bicarbonate and citric acid. Furthermore, floating strength was influenced by the concentration of HPC within the formulation. Granules prepared in this way show good physical characteristics, floating ability, and drug release properties when placed in simulated gastric fluid. Moreover, the drug release and floating properties can be controlled by modification of the ratio or physical characteristics of the excipients used in the formulation.

Preparation and evaluation of sustained-release matrix tablets based on metoprolol and an acrylic carrier using injection moulding.

Sustained-release matrix tablets based on Eudragit RL and RS were manufactured by injection moulding. The influence of process temperature; matrix composition; drug load, plasticizer level; and salt form of metoprolol: tartrate (MPT), fumarate (MPF) and succinate (MPS) on ease of processing and drug release were evaluated. Formulations composed of 70/30% Eudragit RL/MPT showed the fastest drug release, substituting part of Eudragit RL by RS resulted in slower drug release, all following first-order release kinetics. Drug load only affected drug release of matrices composed of Eudragit RS: a higher MPT concentration yielded faster release rates. Adding triethyl citrate enhanced the processability, but was detrimental to long-term stability. The process temperature and plasticizer level had no effect on drug release, whereas metoprolol salt form significantly influenced release properties. The moulded tablets had a low porosity and a smooth surface morphology. A plasticizing effect of MPT, MPS and MPF on Eudragit RS and Eudragit RL was observed via DSC and DMA. Solubility parameter assessment, thermal analysis and X-ray diffraction demonstrated the formation of a solid solution immediately after production, in which H-bonds were formed between metoprolol and Eudragit as evidenced by near-infrared spectroscopy. However, high drug loadings of MPS and MPF showed a tendency to recrystallise during storage. The in vivo performance of injection-moulded tablets was strongly dependent upon drug loading.

The development of an inline Raman spectroscopic analysis method as a quality control tool for hot melt extruded ramipril fixed-dose combination products.

Currently in the pharmaceutical industry, continuous manufacturing is an area of significant interest. In particular, hot-melt extrusion (HME) offers many advantages and has been shown to significantly reduce the number of processing steps relative to a conventional product manufacturing line. To control product quality during HME without process interruption, integration of inline analytical technology is critical. Vibrational spectroscopy (Raman, NIR and FT-IR) is often employed and used for real-time measurements because of the non-destructive and rapid nature of these analytical techniques. However, the establishment of reliable Process Analytical Technology (PAT) tools for HME of thermolabile drugs is challenging. Indeed, the Raman effect is inherently weak and might be subject to interference. Moreover, during HME, heating and photodecomposition can occur and disrupt spectra acquisition. The aim of this research article was to explore the use of inline Raman spectroscopy to characterise a thermolabile drug, ramipril (RMP), during continuous HME processing. Offline measurements by HPLC, LC-MS and Raman spectroscopy were used to characterise RMP and its main degradation product, ramipril-diketopiperazine (RMP-DKP, impurity K). A set of HME experiments together with inline Raman spectroscopic analyses were performed. The feasibility of implementing inline Raman spectroscopic analysis to quantify the level of RMP and RMP-DKP in the extrudate was addressed. Two regions in the Raman spectrum were selected to differentiate RMP and RMP-DKP. When regions were combined, a principle component analysis (PCA) model defined by these two main components (PC 1 = 50.1% and PC 2 = 45%) was established. Using HPLC analyses, we were able to confirm that the PC 1 score was attributed to the level of RMP-DKP, and the PC 2 score was related to the RMP drug content. Investigation of the PCA scatterplot indicated that HME processing temperature was not the only factor causing RMP degradation. Additionally, the plasticiser content, feeding speed and screw rotating speed contributed to RMP degradation during HME processing.

A comparative study between hot-melt extrusion and spray-drying for the manufacture of anti-hypertension compatible monolithic fixed-dose combination products.

The purpose of this work was to investigate the application of different advanced continuous processing techniques (hot melt extrusion and spray drying) to the production of fixed-dose combination (FDC) monolithic systems comprising of hydrochlorothiazide and ramipril for the treatment of hypertension. Identical FDC formulations were manufactured by the two different methods and were characterised using powder X-ray diffraction (PXRD) and modulated differential scanning calorimetry (mDSC). Drug dissolution rates were investigated using a Wood's apparatus, while physical stability was assessed on storage under controlled temperature and humidity conditions. Interestingly both drugs were transformed into their amorphous forms when spray dried, however, hydrochlorothiazide was determined, by PXRD, to be partially crystalline when hot melt extruded with either polymer carrier (Kollidon® VA 64 or Soluplus®). Hot melt extrusion was found to result in significant degradation of ramipril, however, this could be mitigated by the inclusion of the plasticizer, polyethylene glycol 3350, in the formulation and appropriate adjustment of processing temperature. The results of intrinsic dissolution rate studies showed that hot-melt extruded samples were found to release both drugs faster than identical formulations produced via spray drying. However, the differences were attributable to the surface roughness of the compressed discs in the Wood's apparatus, rather than solid state differences between samples. After a 60-day stability study spray dried samples exhibited a greater physical stability than the equivalent hot melt extruded samples.

Protection of mice from fatal bubonic and pneumonic plague by passive immunization with monoclonal antibodies against the F1 protein of Yersinia pestis.

Monoclonal antibodies (MAbs) to the fraction 1 (F1) protein of Yersinia pestis protected mice against fatal pneumonic as well as bubonic plague from wild-type F1+ organisms. The rare isolation of a virulent F1- isolate from surviving animals supports earlier studies suggesting that improved vaccines should consist of immunogens to protect against F1- variants. The high degree of protection with IgG MAb suggests that secretory IgA is not required for protection from pneumonic plague.

The enteric Campylobacter: they are everywhere.

Although Campylobacter spp. are perhaps the most ubiquitous bacterial pathogens known to man, laboratory diagnosis of Campylobacter enteritis is not particularly problematic. The organism's ability to thrive at elevated temperatures facilitates its isolation directly from stool. Enteric infection with Campylobacter spp., despite its invasive nature, is usually self-limiting in the otherwise healthy individual. Management of enteric Campylobacter infection, therefore, is not complex and for most cases simply involves an appropriate rehydration strategy. In addition to the 'tried and true' approaches of good sanitation and thorough cooking of meats to prevent Campylobacter infection, recent advances in vaccine development appear promising.

Antigenic profiling of yersinia pestis infection in the Wyoming coyote (Canis latrans).

Although Yersinia pestis is classified as a "high-virulence" pathogen, some host species are variably susceptible to disease. Coyotes (Canis latrans) exhibit mild, if any, symptoms during infection, but antibody production occurs postinfection. This immune response has been reported to be against the F1 capsule, although little subsequent characterization has been conducted. To further define the nature of coyote humoral immunity to plague, qualitative serology was conducted to assess the antiplague antibody repertoire. Humoral responses to six plasmid-encoded Y. pestis virulence factors were first examined. Of 20 individual immune coyotes, 90% were reactive to at least one other antigen in the panel other than F1. The frequency of reactivity to low calcium response plasmid (pLcr)-encoded Yersinia protein kinase A (YpkA) and Yersinia outer protein D (YopD) was significantly greater than that previously observed in a murine model for plague. Additionally, both V antigen and plasminogen activator were reactive with over half of the serum samples tested. Reactivity to F1 was markedly less frequent in coyotes (35%). Twenty previously tested antibody-negative samples were also examined. While the majority were negative across the panel, 15% were positive for 1-3 non-F1 antigens. In vivo-induced antigen technology employed to identify novel chromosomal genes of Y. pestis that are up-regulated during infection resulted in the identification of five proteins, including a flagellar component (FliP) that was uniquely reactive with the coyote serum compared with immune serum from two other host species. Collectively, these data suggest that humoral immunity to pLcr-encoded antigens and the pesticin plasmid (pPst)-encoded Pla antigen may be relevant to plague resistance in coyotes. The serologic profile of Y. pestis chromosomal antigens up-regulated in vivo specific to C. latrans may provide insight into the differences in the pathogen-host responses during Y. pestis infection.

Identification of Brucella abortus genes in elk (Cervus elaphus) using in vivo-induced antigen technology (IVIAT) reveals novel markers of infection.

Elk in the Greater Yellowstone Area are a major reservoir for brucellosis, which represents an obstacle to eradication of the disease in domestic livestock. Furthermore, immune responses to Brucella abortus infection in the wild host are not well-understood. In this regard, in vivo-induced antigen technology (IVIAT) was employed to identify novel B. abortus antigens expressed during infection in elk. Sera collected from sero-positive Wyoming elk were pooled and absorbed against in vitro-grown cultures of B. abortus. Approximately 35,000 E. coli clones, expressing B. abortus DNA, were then screened by colony immunoblot, yielding ten genes with immuno-reactive products, to include seven proteins secreted beyond the inner membrane. Three products, an outer membrane protein (D15), malate dehydrogenase (Mdh), and an ion transporter (AfuA), were examined by Western blot against individual elk serum samples. Sero-reactivity was significantly more frequent for both Mdh and D15 in naturally infected animals, compared to vaccinated and uninfected elk, indicating that antibody to these two antigens is a predictor of natural infection. Cross-reactivity of all three proteins was next examined with serum samples from confirmed brucellosis-positive cattle. While variable patterns of reactivity were seen with the antigens, the sample group was equivalently reactive to AfuA and Mdh, compared to elk, suggesting that these antigens are commonly expressed during infection in both hosts. We conclude that the application of IVIAT to B. abortus may not only facilitate the identification of serologic markers for brucellosis in elk, but may provide further insight into biological processes of the pathogen in different hosts.

Effect of aluminum hydroxide adjuvant and formaldehyde in the formulation of rPA anthrax vaccine.

The serological response and efficacy of Bacillus anthracis recombinant protective antigen (rPA) vaccines formulated with aluminum hydroxide adjuvant, either with or without formaldehyde, were evaluated in rabbits. Rabbits that had been injected with a single dose of 25 microg of rPA adsorbed to 500 microg of aluminum in aluminum hydroxide gel (Alhydrogel) had a significantly higher quantitative anti-rPA IgG ELISA titers (p<0.0001) and toxin neutralizing antibody (TNA) assay titers (p<0.0001) than rabbits tested at the next lowest concentration of aluminum (158 microg). Rabbits injected with two doses of 50 microg of rPA formulated with 500 microg of aluminum also had significantly higher serological responses, as measured by a quantitative anti-rPA IgG ELISA (p<0.0001) and TNA assay (p<0.0001), than sera from rabbits injected with a rPA vaccine formulated without adjuvant. Short-term protection against an aerosol spore challenge (448 LD(50)), however, was not significantly different between the two groups (12/12 and 11/12, respectively). Rabbits injected with a single dose of 50 microg of rPA formulated with 500 microg of aluminum and 0.2% formaldehyde had significantly higher ELISA (p<0.0001) and TNA assay (p<0.0001) titers than rabbits that had been injected with a rPA vaccine formulated with adjuvant but without formaldehyde. Short-term protection against a 125 LD(50) parenteral spore challenge, however, was not significantly different between the two groups (14/24 and 9/24, respectively; p=0.2476). Under the conditions tested in the rabbit animal model, significantly higher serological responses were observed in rabbits that had been injected with rPA formulated with aluminum hydroxide gel adjuvant and formaldehyde. However, differences in short-term efficacy were not observed.

Duration of protection of rabbits after vaccination with Bacillus anthracis recombinant protective antigen vaccine.

Long-term protection of rabbits that had been vaccinated with two doses of a recombinant protective antigen (rPA) vaccine was examined against an aerosol spore challenge with the Ames isolate of Bacillus anthracis at 6 and 12 months. At 6 months after the primary injection, survival was 74.1% (20/27) with quantitative ELISA titer of 22.3 microg of anti-rPA IgG per millilitre and toxin neutralizing antibody (TNA) assay titer of 332. At 12 months after the primary injection, only 37.5% (9/24) of the rabbits were protected with quantitative ELISA titer of 19.8 microg of anti-rPA IgG per millilitre and TNA assay titer of 286. There was a significant loss of protection (p = 0.0117) and a significant difference in survival curves (p = 0.0157) between the 6- and 12-month groups. When ELISA or TNA assay titer, gender, and challenge dose were entered into a forward logistic regression model, week 26 ELISA titer (p = 0.0236) and week 13 TNA assay titer (p = 0.0147) for the 6-month group, and week 26 ELISA titer (p = 0.0326) and week 8 TNA assay titer (p = 0.0190) for the 12-month group, were significant predictors of survival. Neither gender nor challenge dose were identified as having a statistically significant effect on survival. Booster vaccinations with rPA may be required for the long-term protection of rabbits against anthrax.

Comparative vaccine efficacy of different isoforms of recombinant protective antigen against Bacillus anthracis spore challenge in rabbits.

The next-generation human anthrax vaccine developed by the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) is based upon purified Bacillus anthracis recombinant protective antigen (rPA) adsorbed to aluminum hydroxide adjuvant (Alhydrogel). In addition to being safe, and effective, it is important that such a vaccine be fully characterized. Four major protein isoforms detected in purified rPA by native PAGE during research and development were reduced to two primary isoforms in bulk material produced by an improved process performed under Good Manufacturing Practices (GMP). Analysis of both rPA preparations by a protein-isoaspartyl-methyl-transferase assay (PIMT) revealed the presence of increasing amounts of iso-aspartic acid correlating with isoform content and suggesting deamidation as the source of rPA charge heterogeneity. Additional purification of GMP rPA by anion exchange chromatography separated and enriched the two principal isoforms. The in vitro and in vivo biological activities of each isoform were measured in comparison to the whole GMP preparation. There was no significant difference in the biological activity of each isoform compared to GMP rPA when analyzed in the presence of lethal factor using a macrophage lysis assay. Vaccination with the two individual isoforms revealed no differences in cytotoxicity neutralization antibody titers when compared to the GMP preparation although one isoform induced more anti-PA IgG antibody than the GMP material. Most importantly, each of the two isoforms as well as the whole GMP preparation protected 90-100% of rabbits challenged parenterally with 129 LD50 of B. anthracis Ames spores. The equivalent biological activity and vaccine efficacy of the two isoforms suggests that further processing to separate isoforms is unnecessary for continued testing of this next-generation anthrax vaccine.

Heat inactivation of catalase from Staphylococcus aureus MF-31.

The effects of heat on catalase from Staphylococcus aureus lysates were examined. Catalase activity increased with increasing concentrations of potassium phosphate buffer, when heated at temperatures between 50 and 65 degrees C for 10 min. Inactivation of catalase by NaCl during heating was demonstrated. Extended heating of S. aureus cells at 52 degrees C resulted in a slight decrease in catalase activity of the resultant lysates. This decrease was more pronounced in the presence of salt. Heating at 62 degrees C caused a decrease in catalase activity, but not complete inactivation. These results implicate the combined effects of heat, and NaCl in the inactivation of catalase from S. aureus. The findings are consistent with the hypothesis that H2O2 may accumulate as a result of decreased catalase activity and be responsible for the decreased colony-forming ability of stressed S. aureus.

mxiA of Shigella flexneri 2a, which facilitates export of invasion plasmid antigens, encodes a homolog of the low-calcium-response protein, LcrD, of Yersinia pestis.

The plasmid-encoded invasion plasmid antigen (Ipa) export accessory locus of Shigella flexneri 2a, mxiA, was cloned, and the complete DNA sequence of the gene was determined. The mixA open reading frame was found to encode a polypeptide of 74.03 kDa with a pI of 5.02. A hydropathy analysis of the predicted protein revealed a hydrophilic C terminus and an extremely hydrophobic N terminus without a cleavable signal sequence but with several potential membrane-spanning regions. While a homology search did not reveal any significant relatedness of the mxiA DNA sequence to any known bacterial gene sequences, the derived amino acid sequence of MxiA was found to be highly homologous (68%) to the sequence of the protein encoded by the low-calcium-response locus, lcrD, of Yersinia pestis. The lcrD encodes an inner membrane regulatory protein that has an N-terminal membrane anchor and that is implicated in facilitating the export of Y. pestis outer membrane proteins (G. V. Plano, S. S. Barve, and S. C. Straley, J. Bacteriol. 173:7293-7303, 1991). Congo red binding, HeLa cell invasion, and Ipa excretion were restored in two avirulent mxiA fusion mutants when they were transformed with a cloned copy of the mxiA gene. Furthermore, the expression of the cloned mxiA gene was independent of any vector-specified promoter, suggesting that the transcription of mxiA is driven by its own promoter in this clone. In contrast, the overexpression of mxiA that resulted when it was placed under the control of the lac promoter was found to be deleterious in Escherichia coli. We conclude that mxiA is a homolog of the Y. pestis lcrD locus and may function similarly in S. flexneri, either by directly affecting the excretion of virulence factors or by regulating the expression of export accessory genes.

Catalase activity during the recovery of heat-stressed Staphylococcus aureus MF-31.

Sublethal heating of Staphylococcus aureus produced little loss of catalase activity, but incubation of the injured cells in tryptic soy broth, with or without 10% NaCl added, produced an 85% decrease in catalase activity. Cells recovered in tryptic soy broth demonstrated increases in catalase levels after approximately 5 h, whereas in tryptic soy broth with 10% NaCl the levels remained low for at least 12 h. Thus, the loss of catalase activity during the recovery period was greater than during the heat treatment.

Comparison of microdilution and broth dilution techniques for the susceptibility testing of yeasts to 5-fluorocytosine and amphotericin B.

Microdilution and broth dilution techniques were compared for the susceptibility testing of 50 clinical yeast isolates to 5-fluorocytosine and amphotericin B. Good correlation between methods was obtained with all isolates except Cryptococcus neoformans.

Evaluation of an anti-rPA IgG ELISA for measuring the antibody response in mice.

A recombinant protective antigen (rPA)-based enzyme-linked immunosorbent assay (ELISA) was developed to measure the serological response of female A/J mice after inoculation with the new rPA-based anthrax vaccine. Several fundamental parameters of the ELISA were evaluated: specificity, precision, accuracy, linearity, and stability. Experimental results suggested that the quantitative anti-rPA IgG ELISA could be used to measure antibody levels in female A/J mice and may be useful as a potency assay to monitor consistency of manufacture of a rPA-based vaccine for planned clinical trials.

Effect of Hydrogen Peroxide and Sodium Chloride on Enumeration of Thermally Stressed Cells of Staphylococcus aureus.

Evidence is presented suggesting that the decreased enumeration of heat-stressed Staphylococcus aureus cells on selective media is the result of accumulation of metabolic H2O2. It accumulates due to the decreased activity of catalase caused by the synergistic effects of heat and NaCl. Heated cells enumerated anaerobically on tryptic soy agar (TSA) containing 6.5% NaCl (TSAS 6.5) exhibited a 200-fold increase compared to cells enumerated aerobically on the same medium. The anaerobic counts on TSAS 6.5 were similar to the aerobic counts on TSA. Increases in both death and injury occurred when S. aureus was propagated in tryptic soy broth (TSB) plus 10% NaCl (TSBS) instead of TSB before thermal injury. Addition of catalase to TSA and TSA containing 7.5% NaCl (TSAS) increased the count to approximately the same levels on TSA and TSAS as that found following thermal injury after propagation in TSB. Catalase activity was 12-fold higher in stationary phase cells propagated in TSB than in TSBS. Indirect evidence indicates that toxic levels of H2O2 accumulated rapidly, causing one to two log decreases in enumeration after 30 to 60 min incubation on TSAS.

Two novel virulence loci, mxiA and mxiB, in Shigella flexneri 2a facilitate excretion of invasion plasmid antigens.

A bank of over 4,200 lacZ protein fusions in Shigella flexneri 2a was screened for fusions to temperature-regulated promoters. One mutant, BS260, was completely noninvasive on HeLa cells and mapped to a region on the 220-kb virulence plasmid in which we had previously localized several avirulent temperature-regulated operon fusions (A.E. Hromockyj and A.T. Maurelli, Infect. Immun. 57:2963-2970, 1989). The phenotype of BS260 was similar to that of the previously identified mxi (membrane expression of invasion plasmid antigens) mutants, since it made wild-type intracellular levels of the invasion plasmid antigens (Ipa) but was deficient in the surface expression of IpaB and IpaC. Six kilobases of DNA upstream of the BS260 fusion end joint were cloned, but no temperature-regulated promoter was found, whereas the fusion end joint clone of the noninvasive mxi operon fusion mutant BS226 contained a temperature-regulated promoter. The locus defined by BS260 was designated mxiA, and that defined by BS226 was designated mxiB. Closer analysis of the mxiA and mxiB phenotypes by a cell-free enzyme-linked immunosorbent assay revealed that the mutants failed to excrete IpaB and IpaC into the culture medium, whereas wild-type cells actively released these antigens. Excretion of the ipa polypeptides from wild-type bacteria was confirmed by Western blot analysis of culture supernatants. Protease protection experiments revealed that wild-type S. flexneri 2a actually had much lower levels of surface-exposed IpaB and IpaC relative to those in the total antigen pool. In addition, examination of cellular fractions showed that, although there was no IpaB or IpaC in the outer membrane of BS260 and BS226, the antigens did accumulate in the cytoplasmic membrane. A 76-kDa temperature-regulated polypeptide in wild-type S. flexneri was identified as the putative mxiA gene product. These results strongly suggest that IpaB and IpaC represent truly excreted proteins of S. flexneri and that the mxiA and mxiB loci on the plasmid code for accessory proteins required to facilitate their export through the bacterial outer membrane. These data also suggest that mxiA is part of an operon that specifies additional mxi genes. The products of this operon may constitute a unique multicomponent protein secretion apparatus involved in the transport of Shigella virulence determinants.