Influence of Manufacturing Process on the Microstructure, Stability, and Sensorial Properties of a Topical Ointment Formulation.

The manufacturing process for ointments typically involves a series of heating, cooling, and mixing steps. Precise control of the level of mixing through homogenization and the cooling rate, as well as temperature at different stages, is important in delivering ointments with the desired quality attributes, stability, and performance. In this work, we investigated the influence of typical plant processing conditions on the microstructure, stability, and sensorial properties of a model ointment system through a Design of Experiments (DoE) approach. Homogenization speed at the cooling stage after the addition of the solvent (propylene glycol, PG) was found to be the critical processing parameter that affects stability and the rheological and sensorial properties of the ointment. A lower PG addition temperature was also found to be beneficial. The stabilization of the ointment at a lower PG addition temperature was hypothesized to be due to more effective encapsulation by crystallizing mono- and diglycerides at the lower temperature. The in vitro release profiles were found to be not influenced by the processing parameters, suggesting that for the ointment platform studied, processing affects the microstructure, but the effects do not translate into the release profile, a key performance indicator. Our systematic study represents a Quality-by-Design (QbD) approach to the design of a robust manufacturing process for delivering stable ointments with the desired performance attributes and properties.

Formulation and Skin Permeation of Active-Loaded Lipid Nanoparticles: Evaluation and Screening by Synergizing Molecular Dynamics Simulations and Experiments.

Encapsulation into nanoparticles (NPs) is a potential method to deliver pharmaceutical/cosmetic actives deep into the skin. However, understanding the NP formulations and underlying mechanism of active delivery to skin has scarcely been studied. We report a simulation platform that screens, evaluates, formulates, and provides atomic-resolution interpretation of NP-based formulations, and reveals the active permeation mechanism from NPs to skin. First, three actives, namely, ferulic acid (FA), clotrimazole (CZE), and tretinoin (TTN), and five lipid excipients' (Compritol, Precirol, Geleol, Gelot, Gelucire) combinations were screened by MD simulations for the best pairs. For each suggested pair, the actual active and lipid compositions for the synthesis of stable NP formulations were then obtained by experiments. MD simulations demonstrate that in NP formulations, FA and CZE actives are present at the surface of the NPs, whereas TTN actives are present at both the surface and interior of the NP core. The NP shapes obtained by simulation perfectly match with experiments. For each NP, separate MD simulations illustrate that active-loaded NPs approach the skin surface quickly, and then actives translocate from NP surface to skin surface followed by penetration of NPs through skin. The driving force for the translocation which initiates during the penetration process, is the stronger active-skin interaction compared to active-NP interaction. Permeation free energy indicates spontaneous transfer of actives from solution phase to the surface of the skin bilayer. The free energy barriers are increased in the order of FA < TTN < CZE. Significantly lower diffusions of actives are obtained in the main barrier region compared to bulk, and the average diffusion coefficients of actives are in the same order of magnitude (∼10-6 cm2/s). The estimated permeability coefficients (log P) of actives are mainly governed by free energy barriers. The study would facilitate the development of novel lipid-based NP formulations for personal-care/pharmaceutical applications.

Pembrolizumab-induced Myasthenia Gravis: Literature Review of Ocular Manifestations and a Refractory Case.

Immune checkpoint inhibition is a new and promising therapy approved for the treatment of various malignancies. Pembrolizumab is a potent tumor suppressor that acts by upregulating the immune system to recognize cancer cells which may result in disrupted self-tolerance. We describe a case and perform a literature review of myasthenia gravis with ocular manifestations after treatment with pembrolizumab. Our case had bilateral ptosis refractory to conventional treatment, and she remained functionally blind as a result. The literature review included 28 cases of immune-related myasthenia gravis, and a 30% mortality rate excluding deaths from primary cancer progression was shown. Under half had full symptom resolution (n=13, 46%), and there was no clear correlation between specific management strategies and prognosis. Patients with isolated ocular myasthenia gravis (n=9, 32%) were twice as likely to be symptom-free after treatment compared with generalized myasthenia gravis (75% vs. 39%). Respiratory involvement was associated with twice the mortality rate (60% vs. 33%) and triple the risk of noncomplete symptom resolution (20% vs. 61%). The majority of cases had their pembrolizumab discontinued (n=20, 71%), but 3 were successfully rechallenged by utilizing prophylactic low-dose steroids. Patients with immune-related myasthenia gravis experience increased mortality and morbidity but if steroid-responsive, may benefit from the reintroduction of anti-programmed cell death protein 1 therapy for end-stage malignancy with close monitoring. A high index of clinical suspicion for immune-related adverse effects are critical in an era of rising immunotherapy use.

γδ T Cells in Merkel Cell Carcinomas Have a Proinflammatory Profile Prognostic of Patient Survival.

Merkel cell carcinomas (MCC) are immunogenic skin cancers associated with viral infection or UV mutagenesis. To study T-cell infiltrates in MCC, we analyzed 58 MCC lesions from 39 patients using multiplex-IHC/immunofluorescence (m-IHC/IF). CD4+ or CD8+ T cells comprised the majority of infiltrating T lymphocytes in most tumors. However, almost half of the tumors harbored prominent CD4/CD8 double-negative (DN) T-cell infiltrates (>20% DN T cells), and in 12% of cases, DN T cells represented the majority of T cells. Flow cytometric analysis of single-cell suspensions from fresh tumors identified DN T cells as predominantly Vδ2- γδ T cells. In the context of γδ T-cell inflammation, these cells expressed PD-1 and LAG3, which is consistent with a suppressed or exhausted phenotype, and CD103, which indicates tissue residency. Furthermore, single-cell RNA sequencing (scRNA-seq) identified a transcriptional profile of γδ T cells suggestive of proinflammatory potential. T-cell receptor (TCR) analysis confirmed clonal expansion of Vδ1 and Vδ3 clonotypes, and functional studies using cloned γδ TCRs demonstrated restriction of these for CD1c and MR1 antigen-presenting molecules. On the basis of a 13-gene γδ T-cell signature derived from scRNA-seq analysis, gene-set enrichment on bulk RNA-seq data showed a positive correlation between enrichment scores and DN T-cell infiltrates. An improved disease-specific survival was evident for patients with high enrichment scores, and complete responses to anti-PD-1/PD-L1 treatment were observed in three of four cases with high enrichment scores. Thus, γδ T-cell infiltration may serve as a prognostic biomarker and should be explored for therapeutic interventions.See related Spotlight on p. 600.

Stabilization of ferulic acid in topical gel formulation via nanoencapsulation and pH optimization.

Ferulic acid is a potent anti-oxidant with scientifically proven skin care efficacies. However, instability of this active in the skin care products restricted its wide application in beauty and skin care industries. This study aimed to stabilize ferulic acid in topical hydrogel formulation via nanoencapsulation technique. Ferulic acid loaded nanocapsules were prepared via high pressure homogenization method and physicochemically characterized. Mean particle size of ferulic acid loaded nanocapsules was < 300 nm. TEM and SEM images exhibited spherical particles with smooth surface. DSC and XRD results indicated that ferulic acid was completely dissolved in the lipid matrix of the nanocapsules and remained in amorphous form. Two types of hydrogel formulations containing ferulic acid loaded nanocapsules were prepared: Gel A with pH higher and Gel B with pH lower than pKa of ferulic acid. Cross-polarized microscopic image of the gel formulations did not show presence of any un-encapsulated and un-dissolved crystal. Gel B showed slower and controlled release of ferulic acid than Gel A. Ferulic acid permeation through skin mimic from the gel formulation demonstrated controlled permeation. Color stability of the gel and chemical stability of ferulic acid were very good in Gel B, while poor in Gel A (although significantly better than the gel with un-encapsulated ferulic acid). The result clearly indicates that together with nanoencapsulation, low pH (less than pKa of ferulic acid) of the hydrogel was crucial for both product appearance and chemical stability of ferulic acid. In fact, it has been proved that skin care product with low pH is good for skin as it can maintain skin homeostasis and microbiome. Furthermore, the permeation result suggests that ferulic acid may penetrate into deep skin layers and at the same time avoid systemic circulation. Overall, this low pH hydrogel formulation containing nanoencapsulated ferulic acid demonstrates great promise for commercialization.

18F-FDG PET/CT based spleen to liver ratio associates with clinical outcome to ipilimumab in patients with metastatic melanoma.

BACKGROUND: Immune checkpoint blockade such as ipilimumab and anti-PD1 monoclonal antibodies have significantly improved survival in advanced melanoma. Biomarkers are urgently needed as a majority of patients do not respond, despite treatment-related toxicities. We analysed pre-treatment 18F-fluorodeoxyglucose positron emission tomography/computerised tomography (FDG PET/CT) parameters to assess its correlation with patient outcome. METHODS: This retrospective study evaluated pre-treatment FDG PET/CT scans in a discovery cohort of patients with advanced melanoma treated with ipilimumab or anti-PD1. Pre-treatment scans were assessed for maximum tumoral standardised uptake value (SUVmax), metabolic tumour volume (MTV) and spleen to liver ratio (SLR). Progression-free survival (PFS) and overall survival (OS) were characterised and modelled using univariable and multivariable analyses. Correlation of SLR and OS was validated in an independent cohort. Blood parameters and stored sera of patients from the discovery cohort was analysed to investigate biological correlates with SLR. RESULTS: Of the 90 evaluable patients in the discovery cohort: 50 received ipilimumab monotherapy, 20 received anti-PD1 monotherapy, and 20 patients received ipilimumab followed by anti-PD1 upon disease progression. High SLR > 1.1 was associated with poor PFS (median 1 vs 3 months; HR 3.14, p = 0.008) for patients treated with ipilimumab. High SLR was associated with poor OS after ipilimumab (median 1 vs 21 months; HR 5.83, p = 0.0001); as well as poor OS after first line immunotherapy of either ipilimumab or anti-PD1 (median 1 vs 14 months; HR 3.92, p = 0.003). The association of high SLR and poor OS after ipilimumab was validated in an independent cohort of 110 patients (median 2.3 months versus 11.9 months, HR 3.74). SLR was associated with poor OS in a multi-variable model independent of stage, LDH, absolute lymphocyte count and MTV. CONCLUSIONS: Pre-treatment Spleen to liver ratio (SLR) > 1.1 was associated with poor outcome after ipilimumab in advanced melanoma. This parameter warrants prospective evaluation.

Balancing the Hype with Reality: What Do Patients with Advanced Melanoma Consider When Making the Decision to Have Immunotherapy?

BACKGROUND: Immunotherapy has resulted in unprecedented improvements in survival and maintained quality of life for many patients with advanced melanoma. However, durable responses are observed in only a minority of patients, and severe treatment side effects are experienced by 5%-30%. There are no reliable tests that can differentiate between patients who are likely to respond to immunotherapy and those who will not. Hence, new challenges have arisen as clinicians try to facilitate patients in their decision-making regarding immunotherapy. Furthermore, little is known about the real-world patients' experience and understanding of immunotherapy outside the clinical trial setting. Here, we explore the perspectives of patients undergoing immunotherapy for melanoma and focus on factors that influenced their treatment decision-making. MATERIALS AND METHODS: Twenty-three in-depth semistructured interviews were conducted with patients receiving pembrolizumab for stage IV melanoma at an Australian public cancer hospital. Patients were recruited at a range of time points after commencing therapy, and their experience of treatment was explored. Interviews were audio recorded, transcribed verbatim, coded, and analyzed thematically. RESULTS: Immunotherapy is viewed as a symbol of hope, with high-profile anecdotes reinforcing this perception. Only a minority of patients expressed a good understanding of the likely efficacy and potential treatment side effects. Patients are reliant on their clinicians' recommendation regarding immunotherapy treatment decisions. CONCLUSION: Novel treatments such as immunotherapy provide significant hope for patients. This may influence their preference for immunotherapy over and above the usual considerations of the trade-off between efficacy and toxicity. Careful counsel and individualized patient resources may further facilitate treatment decision-making. IMPLICATIONS FOR PRACTICE: This study highlighted some of the misconceptions held by patients that need to be addressed when discussing the possibility of receiving treatment with immunotherapy for advanced melanoma. Patients placed a lot of importance on high-profile anecdotes rather than truly understanding likely outcomes of treatment based on personal circumstances. The majority of patients had a poor understanding of the potential side effects and long-term implications of treatment with immunotherapy. Careful counsel is required in order to facilitate informed decision-making about treatment and to ensure possible side effects are known and appreciated. Further research is needed to develop tools to aid decision-making in everyday clinical practice.

Outcomes of Australian patients receiving non-funded anti-PD-1 immune checkpoint inhibitors for non-melanoma cancers.

AIM: To characterize the outcomes of patients with nonmelanoma solid tumors receiving anti-PD-1 immunotherapy not funded by the Australian Pharmaceutical Benefits Scheme. METHODS: Medical records of patients with metastatic nonmelanoma tumor diagnoses treated with anti-PD-1 (self-funded pembrolizumab or nivolumab through an access program) from January 1, 2014, to December 31, 2016, at Peter MacCallum Cancer Centre, were retrospectively reviewed. Events after December 31, 2016, were censored. RESULTS: Of 47 patients identified, 27 (57%) had lung cancer. Twenty-six had compassionate access to nivolumab (24 lung, one renal, one gastroesophageal with possible new lung primary). Median overall survival was 5.7 months. Eleven (23%) achieved a partial response; none had complete response. Twenty (43%) had disease progression on first imaging; 16 (48%) of these continued treatment beyond radiological progression, with three achieving subsequent partial responses. Ten (21%) were not re-staged mostly due to rapid deterioration or death. At 6 and 12 months, nine (20%) and two (4%) remained on treatment, respectively. Five (12%) discontinued treatment due to immune-related toxicities. Of 34 patients who died, 71% received treatment within the last month of life; 38% died in an acute hospital. None of 25 patients with poor Eastern Cooperative Oncology Group performance scores of 2-4 responded. CONCLUSION: The response rates and overall survival of patients with NSCLC, renal carcinoma and triple negative breast cancer of good performance status receiving anti-PD-1 therapy outside of a clinical trial are consistent with clinical trial data. However, patients with poor ECOG performance status are unlikely to respond. Careful patient selection and counseling about the potential outcomes of self-funding treatment in this setting is needed.

Evaluating Suspension Formulations of Theophylline Cocrystals With Artificial Sweeteners.

Pharmaceutical cocrystals have garnered significant interest as potential solids to address issues associated with formulation development of drug substances. However, studies concerning the understanding of formulation behavior of cocrystals are still at the nascent stage. We present results of our attempts to evaluate suspension formulations of cocrystals of an antiasthmatic drug, theophylline, with 2 artificial sweeteners. Stability, solubility, drug release, and taste of the suspension formulations were evaluated. Suspension that contained cocrystal with acesulfame showed higher drug release rate, while a cocrystal with saccharin showed a significant reduction in drug release rate. The cocrystal with saccharin was found stable in suspension for over 9 weeks at accelerated test condition; in contrast, the cocrystal with acesulfame was found unstable. Taste analysis using an electronic taste-sensing system revealed improved sweetness of the suspension formulations with cocrystals. Theophylline has a narrow therapeutic index with a short half-life which necessitates frequent dosing. This adversely impacts patient compliance and enhances risk of gastrointestinal and cardiovascular adverse effects. The greater thermodynamic stability, sweetness, and sustained drug release of the suspension formulation of theophylline-saccharin could offer an alternative solution to the short half-life of theophylline and make it a promising formulation for treating asthmatic pediatric and geriatric patients.

Effects of biochar on hydraulic conductivity of compacted kaolin clay.

Compacted clay is widely used as capillary barriers in landfill final cover system. Recently, biochar amended clay (BAC) has been proposed as a sustainable alternative cover material. However, the effects of biochar on saturated hydraulic conductivity (ksat) of clay with high degree of compaction is not yet understood. The present study aims to investigate the effects of biochar on ksat of compacted kaolin clay. Soil specimens were prepared by amending kaolin clay with biochar derived from peanut-shell at 0, 5 and 20% (w/w). The ksat of soil specimens was measured using a flexible water permeameter. The effects of biochar on the microstructure of the compacted clay was also investigated using MIP. Adding 5% and 20% of biochar increased the ksat of compacted kaolin clay from 1.2 × 10-9 to 2.1 × 10-9 and 1.3 × 10-8 ms-1, respectively. The increase in ksat of clay was due to the shift in pore size distribution of compacted biochar-amended clay (BAC). MIP results revealed that adding 20% of biochar shifted the dominant pore diameter of clay from 0.01-0.1 µm (meso- and macropores) to 0.1-4 µm (macropores). Results reported in this communication revealed that biochar application increased the ksat of compacted clay, and the increment was positively correlated to the biochar percentage.

Clinical and palliative care outcomes for patients of poor performance status treated with antiprogrammed death-1 monoclonal antibodies for advanced melanoma.

BACKGROUND: Antiprogrammed death-1 antibodies (anti-PD1) have response rates of 40% in metastatic melanoma. Patients with poor performance status (PS) were excluded from clinical trials, yet use of anti-PD1 is widespread in clinical practice. Literature regarding clinical and palliative care outcomes in patients with poor PS treated with anti-PD1 is lacking. METHODS: Retrospective review of outcomes for all patients with advanced melanoma treated with anti-PD1 between 2012 and June 2015 at Peter MacCallum Cancer Centre, a tertiary specialist cancer center in Australia. RESULTS: Between 2012 and 2015, 91 patients received anti-PD1: median age 63, 65% males, 77% elevated LDH>1xULN (37/48 patients). Fifty-eight patients had baseline ECOG PS of 0-1 (64%), 24 patients ECOG PS 2-3 (26%) and ECOG PS was not recorded in nine patients (10%). Median overall survival (OS) for the ECOG PS 0-1 group was 19.5 months and 1.8 months for ECOG PS 2-3 (HR 5.5; 95% CI, 9.1-50.3; P = 0.0001). Tumor response was 23/58 (39%) in ECOG PS 0-1, 2/16 (12%) in ECOG PS 2 and 0/8 in ECOG PS 3. Toxicity did not differ between different groups. ECOG PS 2-3 patients were more likely to be treated and hospitalized within the last month of life compared to ECOG PS 0-1 patients, RR 1.75 (95% CI, 1.04-2.56, P = 0.019) and RR 1.73 (95% CI, 1.10-2.16, P = 0.009), respectively. ECOG PS 2-3 patients were more likely to die in an acute hospital RR 2.68 (95% CI, 1.17-6.51, P = 0.016). CONCLUSIONS: Patients with poor baseline PS have a significantly lower OS and reduced response to anti-PD1. Further quality of life and palliative care research is needed.

Efficacy of anti-PD-1 therapy in patients with melanoma brain metastases.

BACKGROUND: There is limited data on the efficacy of anti-programmed death 1 (PD-1) antibodies in patients (pts) with melanoma brain metastasis (BM), particularly those which are symptomatic. METHOD: We retrospectively assessed pts with melanoma BM treated with PD-1 antibodies, nivolumab and pembrolizumab. Clinicopathologic and treatment parameters were collected and outcomes determined for intracranial (IC) response rate (RR) using a modified RECIST criteria, with up to five IC target lesions used to determine IC response, disease control rate (DCR) and progression-free survival (PFS). RESULTS: A total of 66 pts were identified with a median follow up of 7.0 months (range 0.8-24.5 months). A total of 68% were male and 45% BRAF V600 mutation positive. At PD-1 antibody commencement, 50% had an elevated LDH; 64% had local therapy to BM prior to commencing anti-PD1, of which 5% had surgical resection, 14% stereotactic radiosurgery (SRS), 18% whole-brain radiotherapy (WBRT), 27% had surgery and radiotherapy. Twenty-one per cent started anti-PD-1 as first line systemic therapy. No pt had prior anti-PD-1 treatment. The IC overall RR was 21 and DCR 56%. Responses occurred in 21% of pts with symptomatic BM. The median OS was 9.9 months (95% CI 6.93-17.74). Pts with symptomatic BM had shorter PFS than those without symptoms (2.7 vs 7.4 months, P=0.035) and numerically shorter OS (5.7 vs 13.0 months, P=0.068). Pts requiring corticosteroids also had a numerically shorter PFS (3.2 vs 7.4 months, P=0.081) and OS (4.8 vs 13.1 months, P=0.039). CONCLUSIONS: IC responses to anti-PD-1 antibodies occur in pts with BM, including those with symptomatic BM requiring corticosteroids. Prospective trials evaluating anti-PD-1 therapy in pts with BM are underway.

The Advantages and Challenges of Using FDG PET/CT for Response Assessment in Melanoma in the Era of Targeted Agents and Immunotherapy.

The treatment of melanoma has been revolutionised in recent years by advances in the understanding of the genomic landscape of this disease, which has led to the development of new targeted therapeutic agents, and the ability to therapeutically manipulate the immune system through inhibition of cancer cell-T-cell interactions that prevent an adaptive immune response. While these therapeutic interventions have dramatically improved the prospects of survival for patients with advanced melanoma, they bring significant complexity to the interpretation of therapeutic response because their mechanisms and temporal profile of response vary considerably. In this review, we discuss the mode of action of these emerging therapies and their toxicities to provide a framework for the use of FDG PET/CT in therapeutic response assessment. We propose that the greatest utility of PET in assessment of response to agents that abrogate signalling related to BRAF mutation is for early assessment of resistance, while in anti-CTLA4 therapy, immunological flare can compromise early assessment of response but can identify potentially life-threatening autoimmune reactions. For anti-PD1/PDL1 therapy, the role of FDG PET/CT is more akin to its use in other solid malignancies undergoing treatment with conventional chemotherapy. However, further research is required to optimise the timing of scans and response criteria in this disease.

CD271 Expression on Patient Melanoma Cells Is Unstable and Unlinked to Tumorigenicity.

The stability of markers that identify cancer cells that propagate disease is important to the outcomes of targeted therapy strategies. In human melanoma, conflicting data exist as to whether hierarchical expression of CD271/p75/NGFR (nerve growth factor receptor) marks cells with enriched tumorigenicity, which would compel their specific targeting in therapy. To test whether these discrepancies relate to differences among groups in assay approaches, we undertook side-by-side testing of published methods of patient-derived melanoma xenografting (PDX), including comparisons of tissue digestion procedures or coinjected Matrigel formulations. We found that CD271(-) and CD271(+) melanoma cells from each of seven patients were similarly tumorigenic, regardless of assay variations. Surprisingly variable CD271 expression patterns were observed in the analyses of sibling PDX tumors (n = 68) grown in the same experiments from either CD271(-) or CD271(+) cells obtained from patients. This indicates unstable intratumoral lineage relationships between CD271(-) and CD271(+) melanoma cells that are inconsistent with classical, epigenetically based theories of disease progression, such as the cancer stem cell and plasticity models. SNP genotyping of pairs of sibling PDX tumors grown from phenotypically identical CD271(-) or CD271(+) cells showed large pairwise differences in copy number (28%-48%). Differences were also apparent in the copy number profiles of CD271(-) and CD271(+) cells purified directly from each of the four melanomas (1.4%-23%). Thus, CD271 expression in patient melanomas is unstable, not consistently linked to increased tumorigenicity and associated with genetic heterogeneity, undermining its use as a marker in clinical studies. Cancer Res; 76(13); 3965-77. ©2016 AACR.

Differential kinetics of response and toxicity using stereotactic radiation and interventional radiological coiling for pulmonary arterio-venous shunting from metastatic leiomyosarcoma.

Case report demonstrating the differential kinetics of response and toxicity using stereotactic radiation and interventional radiological coiling for pulmonary arterio-venous shunting from leiomyosarcoma pulmonary metastases.

Impact of clinical pharmacists in the emergency department of an Australian public hospital: A before and after study.

OBJECTIVE: To evaluate the impact of an ED pharmacy service on ED clinical staff and hospital pharmacist activity. METHODS: A prospective study measuring pharmacist activities and surveying ED staff attitudes and experience before and after commencement of an ED pharmacy service. RESULTS: There were 2275 and 2072 hospital-wide pharmacist occasions of service recorded over a 1 month period before and after implementation of the ED pharmacy service, respectively; 339 (16.4%) of these occurred in the ED post-implementation. ED pharmacists most commonly were involved in obtaining medication histories (74% of ED occasions of service); 43% of all pharmacist-performed medication histories occurred in the ED. Post-implementation of the service, 26% of medication interventions occurred in the ED with the number of medication errors identified by ward pharmacists decreasing by 11%; 59% of ED pharmacist medication interventions were clinically significant. ED clinicians perceived the greatest impact of the service to be on patient education and medication safety. Qualitative feedback was overwhelmingly positive. CONCLUSIONS: Pharmacy staff can rapidly become a vital component of clinical service provision in the ED, contributing to medication safety from the point of patient entry into the hospital and impacting ED clinicians and whole of hospital activity for pharmacists.

Evidence supporting the 19 ß-strand model for Tom40 from cysteine scanning and protease site accessibility studies.

Most proteins found in mitochondria are translated in the cytosol and enter the organelle via the TOM complex (translocase of the outer mitochondrial membrane). Tom40 is the pore forming component of the complex. Although the three-dimensional structure of Tom40 has not been determined, the structure of porin, a related protein, has been shown to be a ß-barrel containing 19 membrane spanning ß-strands and an N-terminal α-helical region. The evolutionary relationship between the two proteins has allowed modeling of Tom40 into a similar structure by several laboratories. However, it has been suggested that the 19-strand porin structure does not represent the native form of the protein. If true, modeling of Tom40 based on the porin structure would also be invalid. We have used substituted cysteine accessibility mapping to identify several potential ß-strands in the Tom40 protein in isolated mitochondria. These data, together with protease accessibility studies, support the 19 ß-strand model for Tom40 with the C-terminal end of the protein localized to the intermembrane space.