Proteolysis of vaginally administered bovine lactoferrin: clearance, inter-subject variability, and implications for clinical dosing.

This report describes proteolytic fragmentation and clearance of bovine lactoferrin (bLF) upon intravaginal administration in premenopausal women. Tablet formulations (MTbLF) containing 300 mg of bLF progressed through three phases: Pre-Dissolution, Dissolution, and Washout, over a 30-h time course. Tablets dissolved slowly, replenishing intact 80 kDa bLF in vaginal fluid (VF) as proteolysis occurred. bLF was initially cleaved approximately in half between its N- and C-lobes, then degraded into sub-fragments and small peptides. The extent of proteolysis was less than 10-20% across multiple subjects. Concentrations of both intact 80 kDa bLF and smaller fragments decreased in VF with a similar time course suggesting washout not proteolysis was the main clearance mechanism. Concentrations of intact and/or nicked 80 kDa bLF peaked between 4 and 8 h after administration and remained above 5 mg/mL for approximately 24 h. Experiments with protease inhibitors in ex vivo VF digests suggested an aspartyl protease was at least partially responsible for bLF cleavage. However, digestion with commercial pepsin or in vivo in the human stomach, demonstrated distinctly different patterns of fragments compared to vaginal proteolysis. Furthermore, the 3.1 kDa antimicrobial peptide lactoferricin B was not detected in VF. This suggests pepsin-like aspartyl proteases are not responsible for vaginal proteolysis of bLF.

Antimicrobial activity of bovine lactoferrin against Gardnerella species clinical isolates.

Gardnerella species play a key role in the development and recurrence of Bacterial Vaginosis (BV), a common imbalance of the vaginal microbiota. Because of the high rates of BV recurrence reported after treatment with standard of care antibiotics, as well as the emergence of antibiotic-resistant BV, the development of alternative treatment approaches is needed. Bovine lactoferrin, a well studied iron-binding glycoprotein with selective antimicrobial activity, may ameliorate vaginal dysbiosis either alone or in combination with antibiotics. The present study evaluated the antimicrobial resistance/susceptibility profile of seventy-one presumptive G. vaginalis clinical isolates to metronidazole and clindamycin. In addition, the in vitro antimicrobial activity of Metrodora Therapeutics bovine Lactoferrin (MTbLF) against the tested clinical isolates, both alone and in combination with metronidazole and clindamycin, was in depth evaluated using defined-iron culture conditions. All 71 presumptive G. vaginalis clinical isolates exhibited resistance to metronidazole, with MIC values greater than 256 µg/ml. Different susceptibility profiles were detected for clindamycin. In detail, the vast majority of the tested strains (45%), exhibiting MIC lower than 2 µg/ml, were considered sensitive; 18 strains (25%) with MIC higher or equal to 8 µg/ml, were classified as resistant, whereas the remaining 21 (30%) were classified as intermediate. MTbLF was tested in culture medium at different concentrations (32, 16, 8, 4, 2, 1, and 0.5 mg/ml) showing ability to inhibit the growth of the tested presumptive G. vaginalis clinical isolates, including those metronidazole-resistant, in a dose-dependent and not in a strain-dependent manner. MTbLF, at concentrations ranging from 32 to 8 mg/ml, exerted a statistically different antimicrobial activity compared with lower concentrations (4, 2, 1, and 0.5 mg/ml). A synergistic effect between MTbLF (8 and 4 mg/ml) and clindamycin was revealed for all the tested strains. When tested in the absence of other sources of iron, MTbLF did not support the growth of the tested presumptive G. vaginalis clinical isolates. Bovine lactoferrin may be a potential candidate to treat Gardnerella species infection.

Characterization of proteolytic degradation products of vaginally administered bovine lactoferrin.

When bovine lactoferrin (bLF) contacts human vaginal fluid (VF) it is subjected to proteolytic degradation. This report describes fragmentation patterns of bLF dosed vaginally in clinical trials or incubated ex vivo with VF. A consensus pattern of fragments was observed in samples from different women. The 80 kDa bLF molecule is initially cleaved between its homologous 40 kDa domains, the N-lobe and C-lobe, and then degraded into sub-fragments and mixtures of small peptides. We characterized this fragmentation process by polyacrylamide gel electrophoresis, western blotting, chromatographic separation, and mass spectral sequence analysis. Common to most VF fragmentation patterns were large amounts of an N-lobe 37 kDa fragment and a C-lobe 43 kDa fragment resulting from a single cleavage following tyrosine 324. Both fragments possessed full sets of iron-ligand amino acids and retained iron-binding ability. In some VF samples, alternative forms of large fragments were found, which like the 37+43 kDa pair, totaled 80 kDa. These included 58+22 kDa, 18+62 kDa, and 16+64 kDa forms. In general, the smaller component was from the N-lobe and the larger from the C-lobe. The 18+62 kDa pair was absent in some VF samples but highly abundant in others. This variability suggests multiple endopeptidases are involved, with the 18 kDa fragment's presence dependent upon the balance of enzymes. Further action of VF endopeptidases produced smaller peptide fragments, and we found evidence that exopeptidases trimmed their N- and C-termini. The 3.1 kDa antimicrobial peptide lactoferricin B was not detected. These studies were facilitated by a novel technique we developed: tricolor western blots, which enabled simultaneous visualization of N- and C-terminal epitopes.

Randomised controlled trial and economic evaluation of a targeted cancer awareness intervention for adults living in deprived areas of the UK.

BACKGROUND: Cancer outcomes are poor in socioeconomically deprived communities, with low symptom awareness contributing to prolonged help-seeking and advanced disease. Targeted cancer awareness interventions require evaluation. METHODS: This is a randomised controlled trial involving adults aged 40+ years recruited in community and healthcare settings in deprived areas of South Yorkshire and South-East Wales. INTERVENTION: personalised behavioural advice facilitated by a trained lay advisor. CONTROL: usual care. Follow-up at two weeks and six months post-randomisation. PRIMARY OUTCOME: total cancer symptom recognition score two weeks post-randomisation. RESULTS: Two hundred and thirty-four participants were randomised. The difference in total symptom recognition at two weeks [adjusted mean difference (AMD) 0.6, 95% CI: -0.03, 1.17, p = 0.06] was not statistically significant. Intervention participants reported increased symptom recognition (AMD 0.8, 95% CI: 0.18, 1.37, p = 0.01) and earlier intended presentation (AMD -2.0, 95% CI: -3.02, -0.91, p < 0.001) at six months. "Lesser known" symptom recognition was higher in the intervention arm (2 weeks AMD 0.5, 95% CI: 0.03, 0.97 and six months AMD 0.7, 95% CI: 0.16, 1.17). Implementation cost per participant was £91.34, with no significant between-group differences in healthcare resource use post-intervention. CONCLUSIONS: Improved symptom recognition and earlier anticipated presentation occurred at longer-term follow-up. The ABACus Health Check is a viable low-cost intervention to increase cancer awareness in socioeconomically deprived communities. CLINICAL TRIAL REGISTRATION: ISRCTN16872545.

Recruitment and retention of participants from socioeconomically deprived communities: lessons from the Awareness and Beliefs About Cancer (ABACus3) Randomised Controlled Trial.

BACKGROUND: Recruitment of research participants poses challenges in socioeconomically deprived areas. The Awareness and Beliefs About Cancer (ABACus) phase 3 Randomised Control Trial recruited adult participants from socioeconomically deprived areas using a combined healthcare/community engagement model. We report the strategies used to successfully recruit and retain our trial participant sample. METHODS: Community and healthcare settings in areas of high socioeconomic deprivation were identified by lay advisors who recruited participants opportunistically or by appointment. Follow-up was done by telephone or post at 2-weeks and 6-months after recruitment, and all participants were offered financial incentives. Qualitative interviews were conducted with lay advisors regarding their experience and reflections. RESULTS: The lay advisors identified and contacted 107 potential recruitment venues across South and West Yorkshire and South East Wales of which 41.1% (n = 42) were opened for recruitment. A total of 234 participants were recruited, with 91% (n = 212) retention at 2-weeks and 85% (n = 199) at 6-months. Community settings yielded 75% (n = 176) of participants. Participants had a mean age of 61.3 years and 63.3% (n = 148) were female, with 66% (n = 154) resident in the most deprived geographical areas. Lay advisors described recruitment as intensive, although engaging participants was easier in community settings. CONCLUSIONS: The ABACus3 trial achieved recruitment and high retention with a population that is often "hard to reach" or entirely missed in health research. Strategies were specifically tailored to engage the venues and adult residents of highly deprived areas. Future studies recruiting adults living in the most deprived areas might benefit from community recruitment and from collaborating with local gatekeepers who are key to engagement. This study adheres to CONSORT guidelines. TRIAL REGISTRATION: Retrospectively registered with ISRCTN ( http://www.isrctn.com/ISRCTN16872545 ) on 12.01.2018.

Improving cancer symptom awareness and help-seeking among adults living in socioeconomically deprived communities in the UK using a facilitated health check: A protocol for the Awareness and Beliefs About Cancer (ABACus) Randomised Control Trial.

BACKGROUND: Cancer survival is lower in socioeconomically deprived communities, partly due to low awareness of symptoms, negative beliefs and delayed help-seeking. We developed an interactive health check questionnaire facilitated by trained lay advisors. It entails 29 questions about background, lifestyle and health with tailored behaviour change advice. Personalised results are printed using a traffic light (red/amber/green) system, highlighting areas where action should be taken. This is an individually randomised control trial to test effectiveness of the health check on symptom recognition. METHODS: A total 246 participants aged 40+ years will be recruited from community and healthcare settings in socioeconomically deprived areas of Yorkshire and South Wales. Participants will be randomised to receive the health check or standard care (1:1 ratio). Outcome measures include: adapted Awareness and Beliefs about Cancer (primary outcome), brief State Trait Anxiety Inventory, intentions and motivation to adopt recommended health behaviours (early symptom presentation, cancer screening and lifestyle behaviours), adapted Client Service Receipt Inventory, brief medical history/screening and demographic questionnaire at: baseline; 2-weeks; and 6-months post-randomisation. A purposive sample of intervention sessions will be audio-recorded (n = 24) and half will additionally be observed (n = 12). Semi-structured interviews will take place at 2-weeks (n = 30) and 6-months (n = 15-20) post-randomisation. The primary analysis will compare cancer symptom recognition scores between arms at 2-weeks. Secondary analysis will assess cancer beliefs, barriers/time to presentation, screening and lifestyle behaviours, anxiety and costs. A process evaluation will assess intervention fidelity, dose and contamination. The London-Surrey NHS Research Ethics Committee (Ref: 17/LO/1507) approved this trial. DISCUSSION: This is a trial of a theoretically underpinned complex intervention which has undergone phase 1 and 2 development work. The findings will evaluate evidence about the effect of the health check on symptom awareness. Although there are few exclusion criteria there are limitations regarding the population we are able to reach, who may have even higher risks of late diagnosis and poor cancer prognosis. However, the health check has the potential to improve cancer symptom awareness and encourage early help-seeking behaviour in deprived populations, thereby reducing inequalities in longer term cancer outcomes. TRIAL REGISTRATION: Retrospectively registered with ISRCTN (Ref: ISRCTN16872545 ) on 12.01.2018.

The case for targeting community pharmacy-led health improvement: Findings from a skin cancer campaign in Wales.

UNLABELLED: The use of community pharmacies to deliver health improvement campaigns is well established. Cancer incidence is closely related to increasing levels of deprivation. Because community pharmacies are more prevalent in deprived areas there is potential for them to make an important contribution to health improvement by delivering interventions aimed at reducing cancer incidence amongst those at greatest risk. OBJECTIVES: The aims of the study were: to examine the association between high risk behaviour and deprivation and contribute evidence to the case for or against targeting cancer prevention campaigns at specific risky behaviour in areas of high deprivation. METHODS: This study has an ecological design and involved retrospective analysis of data derived from 5739 sun-safety quizzes completed by pharmacy users at 714 community pharmacies in Wales during May 2014. RESULTS: Levels of participation in the campaign and high risk sun-safety behaviours were higher in more deprived areas. Respondents from deprived areas had significantly lower sun-safety knowledge. 3802 (66.2%) respondents reported 'excellent' or 'good' knowledge of behaviours which promote sun-safety but this did not necessarily translate into how individuals behaved. 3787 (66.1%) respondents considered community pharmacies an acceptable location for the campaign and for discussing the signs and symptoms of skin cancer. KEY FINDINGS: Results show association between high risk behaviour and geographically defined deprivation adding to the case for targeting cancer prevention campaigns at specific behaviours and geographies. CONCLUSIONS: Community pharmacies appear to be acceptable locations from which to deliver health improvement campaigns in terms of participant recruitment, ease of delivery, and pharmacy user feedback.

TLR8 stimulation enhances cetuximab-mediated natural killer cell lysis of head and neck cancer cells and dendritic cell cross-priming of EGFR-specific CD8+ T cells.

BACKGROUND: Cetuximab is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody that prolongs survival in the treatment for head and neck cancer (HNC), but only in 10-20 % of patients. An immunological mechanism of action such as natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) has been suggested. We investigated the effects of activating toll-like receptor (TLR)-8 to enhance activity of cetuximab-stimulated, FcγR-bearing cells. OBJECTIVE: To determine the capability of TLR8-stimulation to enhance the activation and function of NK cells and dendritic cells (DC) in the presence of cetuximab-coated HNC cells. METHODS: Peripheral blood mononuclear cells (PBMC), NK, DC, and CD8(+) T cells were isolated and analyzed using (51)Cr release ADCC, flow cytometry analysis, cytokine ELISA, and EGFR853-861 tetramer staining. RESULTS: TLR8 stimulation of unfractionated PBMC led to enhanced cetuximab-mediated ADCC in healthy donors (p < 0.01) and HNC patients (p < 0.001), which was dependent on NK cells. Secretion of Th1 cytokines TNFα (p < 0.0001), IFNγ (p < 0.0001), and IL-12p40 (p < 0.005) was increased. TLR8 stimulation of PBMC augmented cetuximab-enhanced NK cell degranulation (p < 0.001). TLR8-stimulated NK cells enhanced DC maturation markers CD80, CD83, and CD86 in co-culture with cetuximab-treated HNC cells. TLR8 stimulation of NK-DC co-cultures significantly increased DC priming of EGFR-specific CD8(+) T cells in the presence of cetuximab. DISCUSSION: VTX-2337 and cetuximab combination therapy can activate innate and adaptive anti-cancer immune responses. Further investigation in human trials will be important for determining the clinical benefit of this combination and for determining biomarkers of response.

The ultra-potent and selective TLR8 agonist VTX-294 activates human newborn and adult leukocytes.

BACKGROUND: Newborns display distinct immune responses that contribute to susceptibility to infection and reduced vaccine responses. Toll-like receptor (TLR) agonists may serve as vaccine adjuvants, when given individually or in combination, but responses of neonatal leukocytes to many TLR agonists are diminished. TLR8 agonists are more effective than other TLR agonists in activating human neonatal leukocytes in vitro, but little is known about whether different TLR8 agonists may distinctly activate neonatal leukocytes. We characterized the in vitro immuno-stimulatory activities of a novel benzazepine TLR8 agonist, VTX-294, in comparison to imidazoquinolines that activate TLR8 (R-848; (TLR7/8) CL075; (TLR8/7)), with respect to activation of human newborn and adult leukocytes. Effects of VTX-294 and R-848 in combination with monophosphoryl lipid A (MPLA; TLR4) were also assessed. METHODS: TLR agonist specificity was assessed using TLR-transfected HEK293 cells expressing a NF-κB reporter gene. TLR agonist-induced cytokine production was measured in human newborn cord and adult peripheral blood using ELISA and multiplex assays. Newborn and adult monocytes were differentiated into monocyte-derived dendritic cells (MoDCs) and TLR agonist-induced activation assessed by cytokine production (ELISA) and co-stimulatory molecule expression (flow cytometry). RESULTS: VTX-294 was ≈ 100x more active on TLR8- than TLR7-transfected HEK cells (EC50, ≈ 50 nM vs. ≈ 5700 nM). VTX-294-induced TNF and IL-1ß production were comparable in newborn cord and adult peripheral blood, while VTX-294 was 1 log more potent in inducing TNF and IL-1ß production than MPLA, R848 or CL075. Combination of VTX-294 and MPLA induced greater blood TNF and IL-1ß responses than combination of R-848 and MPLA. VTX-294 also potently induced expression of cytokines and co-stimulatory molecules HLA-DR and CD86 in human newborn MoDCs. CONCLUSIONS: VTX-294 is a novel ultra-potent TLR8 agonist that activates newborn and adult leukocytes and is a candidate vaccine adjuvant in both early life and adulthood.

VTX-2337 is a novel TLR8 agonist that activates NK cells and augments ADCC.

PURPOSE: We aim to characterize VTX-2337, a novel Toll-like receptor (TLR) 8 agonist in clinical development, and investigate its potential to improve monoclonal antibody-based immunotherapy that includes the activation of natural killer (NK) cells. EXPERIMENTAL DESIGN: HEK-TLR transfectants were used to compare the selectivity and potency of VTX-2337, imiquimod, CpG ODN2006, and CL075. The ability of VTX-2337 to induce cytokine and chemokine production from human peripheral blood mononuclear cells (PBMC) and activation of specific immune cell subsets was examined. The potential for VTX-2337 to activate NK cell activity through direct and indirect mechanisms was also investigated. Finally, we tested the potential for VTX-2337 to augment antibody-dependent cell-mediated cytotoxicity (ADCC), especially in individuals with low-affinity FcγR3A single-nucleotide polymorphism (SNP). RESULTS: VTX-2337 selectively activates TLR8 with an EC(50) of about 100 nmol/L and stimulates production of TNFα and interleukin (IL)-12 from monocytes and myeloid dendritic cells (mDC). VTX-2337 stimulates IFNγ production from NK cells and increases the cytotoxicity of NK cells against K562 and ADCC by rituximab and trastuzumab. Effects of VTX-2337 on NK cells were, in part, from direct activation as increased IFNγ production and cytotoxic activity were seen with purified NK cells. Finally, VTX-2337 augments ADCC by rituximab in PBMCs with different FcγR3A genotypes (V/V, V/F, and F/F at position 158). CONCLUSIONS: VTX-2337 is a novel small-molecule TLR8 agonist that activates monocytes, DCs, and NK cells. Through the activation of NK cells, it has the potential to augment the effectiveness of monoclonal antibody treatments where a polymorphism in FcγR3A limits clinical efficacy.