Carboranes as Potent Phenyl Mimetics: A Comparative Study on the Reversal of ABCG2-Mediated Drug Resistance by Carboranylquinazolines and Their Organic Isosteres.

Multidrug resistance is a major challenge in clinical cancer therapy. In particular, overexpression of certain ATP-binding cassette (ABC) transporter proteins, like the efflux transporter ABCG2, also known as breast cancer resistance protein (BCRP), has been associated with the development of resistance to applied chemotherapeutic agents in cancer therapies, and therefore targeted inhibition of BCRP-mediated transport might lead to reversal of this (multidrug) resistance (MDR). In a previous study, we have described the introduction of a boron-carbon cluster, namely closo-dicarbadodecaborane or carborane, as an inorganic pharmacophore into a polymethoxylated 2-phenylquinazolin-4-amine backbone. In this work, the scope was extended to the corresponding amide derivatives. As most of the amide derivatives suffered from poor solubility, only the amide derivative QCe and the two amine derivatives DMQCc and DMQCd were further investigated. Carboranes are often considered as sterically demanding phenyl mimetics or isosteres. Therefore, the organic phenyl and sterically demanding adamantyl analogues of the most promising carborane derivatives were also investigated. The studies showed that the previously described DMQCd, a penta-methoxylated N-carboranyl-2-phenylquinazolin-4-amine, was by far superior to its organic analogues in terms of cytotoxicity, inhibition of the human ABCG2 transporter, as well as the ability to reverse BCRP-mediated mitoxantrone resistance in MDCKII-hABCG2 and HT29 colon cancer cells. Our results indicate that DMQCd is a promising candidate for further in vitro as well as in vivo studies in combination therapy for ABCG2-overexpressing cancers.

Carborane-Based ABCG2-Inhibitors Sensitize ABC-(Over)Expressing Cancer Cell Lines for Doxorubicin and Cisplatin.

The ABCG2 transporter protein, as part of several known mechanisms involved in multidrug resistance, has the ability to transport a broad spectrum of substrates out of the cell and is, therefore, considered as a potential target to improve cancer therapies or as an approach to combat drug resistance in cancer. We have previously reported carborane-functionalized quinazoline derivatives as potent inhibitors of human ABCG2 which effectively reversed breast cancer resistance protein (BCRP)-mediated mitoxantrone resistance. In this work, we present the evaluation of our most promising carboranyl BCRP inhibitors regarding their toxicity towards ABCG2-expressing cancer cell lines (MCF-7, doxorubicin-resistant MCF-7 or MCF-7 Doxo, HT29, and SW480) and, consequently, with the co-administration of an inhibitor and therapeutic agent, their ability to increase the efficacy of therapeutics with the successful inhibition of ABCG2. The results obtained revealed synergistic effects of several inhibitors in combination with doxorubicin or cisplatin. Compounds DMQCa, DMQCc, and DMQCd showed a decrease in IC50 value in ABCB1- and ABCG2-expressing SW480 cells, suggesting a possible targeting of both transporters. In an HT29 cell line, with the highest expression of ABCG2 among the tested cell lines, using co-treatment of doxorubicin and DMQCd, the effective inhibitory concentration of the antineoplastic agent could be reduced by half. Interestingly, co-treatment of compound QCe with cisplatin, which is not an ABCG2 substrate, showed synergistic effects in MCF-7 Doxo and HT29 cells (IC50 values halved or reduced by 20%, respectively). However, a literature-known upregulation of cisplatin-effluxing ABC transporters and their effective inhibition by the carborane derivatives emerges as a possible reason.

Sweet Battle of the Epimers─Continued Exploration of Monosaccharide-Derived Delivery Agents for Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is a cancer therapy in which boron delivery agents play a crucial role. In theory, delivery agents with high tumor targeting capabilities can lead to selective eradication of tumor cells without causing harmful side effects. We have been working on a GLUT1-targeting strategy to BNCT for a number of years and found multiple promising hit compounds which outperform the clinically employed boron delivery agents in vitro. Herein, we continue our work in the field by further diversification of the carbohydrate scaffold in order to map the optimal stereochemistry of the carbohydrate core. In the sweet battle of the epimers, carborane-bearing d-galactose, d-mannose, and d-allose are synthesized and subjected to in vitro profiling studies─with earlier work on d-glucose serving as the reference. We find that all of the monosaccharide delivery agents display a significantly improved boron delivery capacity over the delivery agents approved for clinical use in vitro, thus providing a sound foundation for advancing toward in vivo preclinical assessment studies.

2-Carboranylquinazoline: The Path to an ABCG2 Inhibitor.

The role of ATP-binding cassette (ABC) transporter-mediated multidrug resistance (MDR) in anti-cancer therapy is often challenging, frequently leading to inefficiency of treatments. Cancer cells exploit efflux transporters, like the breast cancer resistance protein (BCRP, ABCG2), to secrete chemotherapeutic substances. In this study, an N-phenyl-2-carboranylquinazolin-4-amine (8) was designed as inorganic-organic hybrid BCRP inhibitor. In particular, the ABCG2-transporter inhibitor-prominent scaffold N-phenylquinazolin-4-amine was combined with a boron-carbon cluster (carborane) moiety. Introducing a carborane at 2-position of the quinazoline scaffold resulted in an increased inhibitory activity towards human ABCG2 (hABCG2) compared to its recently published regioisomer N-carboranyl-2-phenyl-quinazolin-4-amine. The carboranylquinazoline 8 further showed the ability to reverse hABCG2-mediated drug resistance in MDCKII-hABCG2 cells by lowering the IC50 value of the BCRP-substrate mitoxantrone, similar to the standard reference and strong inhibitor Ko143, without exhibiting intrinsic toxicity in the lower micromolar ranges. These results make compound 8 a promising scaffold for the design of further BCRP inhibitors.

The More the Better-Investigation of Polymethoxylated N-Carboranyl Quinazolines as Novel Hybrid Breast Cancer Resistance Protein Inhibitors.

The ineffectiveness and failing of chemotherapeutic treatments are often associated with multidrug resistance (MDR). MDR is primarily linked to the overexpression of ATP-binding cassette (ABC) transporter proteins in cancer cells. ABCG2 (ATP-binding cassette subfamily G member 2, also known as the breast cancer resistance protein (BCRP)) mediates MDR by an increased drug efflux from the cancer cells. Therefore, the inhibition of ABCG2 activity during chemotherapy ought to improve the efficacy of the administered anti-cancer agents by reversing MDR or by enhancing the agents' pharmacokinetic properties. Significant efforts have been made to develop novel, powerful, selective, and non-toxic inhibitors of BCRP. However, thus far the clinical relevance of BCRP-selective MDR-reversal has been unsuccessful, due to either adverse drug reactions or significant toxicities in vivo. We here report a facile access towards carboranyl quinazoline-based inhibitors of ABCG2. We determined the influence of different methoxy-substitution patterns on the 2-phenylquinazoline scaffold in combination with the beneficial properties of an incorporated inorganic carborane moiety. A series of eight compounds was synthesized and their inhibitory effect on the ABCG2-mediated Hoechst transport was evaluated. Molecular docking studies were performed to better understand the structure-protein interactions of the novel inhibitors, exhibiting putative binding modes within the inner binding site. Further, the most potent, non-toxic compounds were investigated for their potential to reverse ABCG2-mediated mitoxantrone (MXN) resistance. Of these five evaluated compounds, N-(closo-1,7-dicarbadodecaboran(12)-9-yl)-6,7-dimethoxy-2-(3,4,5-trimethoxyphenyl)-quinazolin-4-amine (DMQCd) exhibited the strongest inhibitory effect towards ABCG2 in the lower nanomolar ranges. Additionally, DMQCd was able to reverse BCRP-mediated MDR, making it a promising candidate for further research on hybrid inorganic-organic compounds.

Synthesis and In Vitro Evaluation of a Set of 6-Deoxy-6-thio-carboranyl d-Glucoconjugates Shed Light on the Substrate Specificity of the GLUT1 Transporter.

Glucose- and sodium-dependent glucose transporters (GLUTs and SGLTs) play vital roles in human biology. Of the 14 GLUTs and 12 SGLTs, the GLUT1 transporter has gained the most widespread recognition because GLUT1 is overexpressed in several cancers and is a clinically valid therapeutic target. We have been pursuing a GLUT1-targeting approach in boron neutron capture therapy (BNCT). Here, we report on surprising findings encountered with a set of 6-deoxy-6-thio-carboranyl d-glucoconjugates. In more detail, we show that even subtle structural changes in the carborane cluster, and the linker, may significantly reduce the delivery capacity of GLUT1-based boron carriers. In addition to providing new insights on the substrate specificity of this important transporter, we reach a fresh perspective on the boundaries within which a GLUT1-targeting approach in BNCT can be further refined.

Saliva diagnostics in patients suffering from bisphosphonate-associated osteonecrosis of the jaw: Results of an observational study.

OBJECTIVE: One of the severe side effects of bisphosphonate (BP) therapy is bisphosphonate-associated osteonecrosis of the jaw (BONJ). However, there is no information available about its pathogenesis. Hence, the aim of this observational study was to contribute to discerning this pathogenesis by comparing salivary quantity and quality in patients with BONJ and undergoing BP treatment. MATERIALS AND METHODS: This study included 60 patients divided into three groups. The first group consisted of 20 patients with established BONJ, the second group had 20 patients undergoing BP treatment, and the third group comprised 20 healthy individuals. These groups were analysed for the flow rate of stimulated saliva, buffer capacity, and salivary pH level. RESULTS: Reduced salivation was observed in a significantly high number of patients with established BONJ (n = 8) and those undergoing BP treatment (n = 9) in comparison with the healthy control group (n = 4; p = 0.039). Though the distribution of the mean value of stimulated saliva flow rates in patients undergoing BP treatment was lower than in the control group, the difference was not significant. Moreover, there were no significant differences in the salivary pH level and buffer capacity in patients undergoing BP treatment as compared with the healthy control group. CONCLUSIONS: It is possible that the quantity of human saliva is affected by BP treatment. This reduction in saliva production could have a negative effect on mucosal health and is perhaps a cofactor in the pathogenesis of BONJ.

New keys for old locks: carborane-containing drugs as platforms for mechanism-based therapies.

Icosahedral carboranes in medicine are still an emerging class of compounds with potential beneficial applications in drug design. These highly hydrophobic clusters are potential "new keys for old locks" which open up an exciting field of research for well-known, but challenging important therapeutic substrates, as demonstrated by the numerous examples discussed in this review.

Do measurements of inflammatory mediators in blood predict recurrence in patients with bisphosphonate-related osteonecrosis of the jaws?

Bisphosphonate-related osteonecrosis of the jaws (BRONJ) is well-recognised, difficult to manage, and often recurs. The aim of this study was to examine the value of preoperative measurements of inflammatory mediators in blood in 212 patients with BRONJ who were studied prospectively. Multiple logistic regression analysis was used to assess the importance of the amounts of substances in the blood that are independently associated with the dependent variable "recurrence of BRONJ". The only factor that significantly influenced the development of recurrent BRONJ was reduction in the white cell count (p<0.0001; hazard ratio 5.324; 95% CI 2.373 to 11.945). Neither white cell counts nor C-reactive protein concentrations within or above the reference ranges were significantly associated with recurrent BRONJ. Patients whose white cell counts were lower than the reference range were at increased risk of recurrent BRONJ. This may be a marker of reduced immunocompetence, and additional prophylactic measures or treatment should be considered for these patients.

Sinus floor augmentation with autogenous bone vs. a bovine-derived xenograft - a 5-year retrospective study.

OBJECTIVES: The long-term outcome after sinus augmentation with autogenous bone or a bovine xenograft (Bio-Oss(®)) was assessed in 47 patients. Inclusion criterion was a vertical dimension of the maxilla of <4 mm. After a functional loading period of 60 months, implant survival and reduction in the augmentation height were compared between the two groups evaluated. MATERIAL AND METHODS: Sinus augmentation was performed using mandibular bone grafts or Bio-Oss(®). In the autogenous bone group, 70 implants were placed in 23 patients, while in the Bio-Oss(®) group, 24 patients received 98 implants. Fisher's exact test and equivalence testing were used to compare implant survival rates. RESULTS: The overall survival rate of the implants was 95.8% 5 years after implant insertion. In the autogenous bone group, the implants had a survival rate of 97.1%, while in the Bio-Oss(®) group, 94.9% of the implants survived. The difference was not statistically significant (P > 0.05); both treatments are equivalent (confidence interval 90%) for the equivalence interval [-0.1; 0.1]. 43.5% of the cases showed no reduction in the augmentation height 5 years after implant insertion, when augmentation was performed with autogenous bone, while in the Bio-Oss(®) group, no resorption was found in 50% of the augmented areas. Up to 25% reduction in augmentation height was found in 47.8% in the autogenous and in 45.8% in the Bio-Oss(®) group. In 8.7% of all cases in the autogenous bone group and in 4.2 % in the Bio-Oss(®) group, up to 50% of the augmented height was resorbed. CONCLUSION: After a 5 years evaluation period, Bio-Oss(®) as material for the indication maxillary sinus augmentation shows to be equivalent to autogenous bone grafting.

Immunohistological expression of human ß-defensin-1 and human ß-defensin-2 in exacerbation of acute and secondary chronic osteomyelitis of the mandible.

BACKGROUND: The majority of patients diagnosed with osteomyelitis of the jaw have severe complaints. Unfortunately, the pathogenesis still remains unclear. Human ß-defensins expressed in epithelial and bone tissues as a part of the innate immunity may be involved in disease development. In this study, we hypothesize that expression levels of human ß-defensin-1 and -2 in the acute and secondary chronic osteomyelitis may be altered in comparison with healthy bone and with bisphosphonate-associated necrosis as well as irradiation from a previous study. METHODS: Bone samples were collected during surgical debridement in a total of eight patients suffering from acute or secondary chronic osteomyelitis of the jaw. Expression levels of hBD-1 and -2 were quantified and related to non-stained cells. Ratios were compared by one-way ANOVA and multiple tests by Holm-Bonferroni. RESULTS: Multiple testing revealed no significant differences for expression levels of human ß-defensin-1 between all groups, whereas labeling index of human ß-defensin-2 was significantly different between specimens of bisphosphonate-associated osteonecrosis of the jaws and all other groups. No significant difference occurred between samples of floride osteomyelitis and healthy bone for expression of hBD-1 and -2. CONCLUSIONS: Although the affected patients showed all clinical signs of acute inflammation, expression levels in acute and secondary chronic osteomyelitis in the jaws did not reveal statistically significant differences compared with healthy bone samples. The weak immunological host response in terms of a putative genetically predisposition should be further discussed as pathogenesis factor for osteomyelitis in the future.

Osteo-radio-necrosis (ORN) and bisphosphonate-related osteonecrosis of the jaws (BRONJ): the histopathological differences under the clinical similarities.

OBJECTIVES: Both osteoradionecrosis (ORN) and bisphosphonate associated osteonecrosis of the jaws (BRONJ) present clinically as regions of exposed necrotic bone. The study aimed to demonstrate the histopathological differences behind the observed clinical similarities. STUDY DESIGN: Ten ORN specimens and ten BRONJ specimens were used, as well as ten samples of normal mandibular bone as control. Two bone-specific stainings were used, i.e. Sirius Red for the study of the relative presence of collagen types I and III and toluidine blue for the study the osteon density. RESULTS: The Red Green Blue (RGB)-analysis of the specimens stained with Sirius Red identified significant differences between the chromatic patterns observed in bone preparations of patients suffering from ORN when compared to both BRONJ and control samples. Moreover, the osteon density of the BRONJ samples was significantly lower when compared to ORN and normal bone samples. CONCLUSIONS: The demonstrated differences in the bone architecture and in the bone collagen content between the two pathological conditions most likely reflect underlying pathophysiological differences.

Osteonecrosis of the jaw in a Crohn's disease patient following a course of Bisphosphonate and Adalimumab therapy: a case report.

BACKGROUND: Bisphosphonates have a widespread indication for osteoporosis and are also applied in cancer patients with skeletal-related conditions. Bisphosphonate-associated osteonecrosis of the jaw (BRONJ) is a feared side effect which is hard to treat and often affects patient's quality of life in an extensive manner. Adalimumab (Humira®), a fully human recombinant antibody specific for tumor necrosis factor- α, is approved for treatment in patients with Inflammatory Bowel Disease like ulcerative colitis or Crohn's disease. CASE PRESENTATION: In March 2013, a 36-year-old female presented with right-sided perimandibular swelling, recurrent facial pain and exposed necrotic bone after previous extraction of tooth 47. She had the medical history of Crohn's disease for more than one decade with chronic active enterocolitis, fistula disease as well as previous oral manifestation and was currently treated with Adalimumab since September 2008. Due to steroid-induced osteoporosis, diagnosed in 2004, she received oral Bisphosphonates (Risedronate) from 2004 until 2007 followed by two infusions of Zoledronic acid in 2008 and 2009. CONCLUSION: This patient with a medical history of Crohn's disease and gastrointestinal remission under Adalimumab therapy presented with osteonecrosis of the jaw after suspended oral and intravenous Bisphosphonate therapy implicating that the biologic therapy with an anti-TNF-α antibody might promote the manifestation of osteonecrosis and compromise oral healing capacity.

The effect of load on heat production, thermal effects and expenditure of time during implant site preparation - an experimental ex vivo comparison between piezosurgery and conventional drilling.

OBJECTIVES: Piezoelectric surgery (PS) is meant to be a gentle osteotomy method. The aim of this study was to compare piezosurgical vs. conventional drilling methods for implant site preparation (ISP) - focusing on load-dependent thermal effect on hard tissue and the expenditure of ISP time. MATERIALS AND METHODS: Three hundred and sixty ISP were performed on ex vivo pig heads using piezosurgery, spiral burs (SB) and trephine burs (TB). The load applied was increased from 0 to 1000 g in 100-g intervals. Temperature within the bone was measured with a thermocouple, and duration was recorded with a stop watch. Thermal effects were histomorphometrically analysed. Twelve ISPs per technique were performed at the lateral wall of the maxillary sinus. RESULTS: PS yields the highest mean temperatures (48.6 ± 3.4°C) and thermal effects (200.7 ± 44.4 µm), both at 900-1000 g. Duration is reduced with a plus of load and significantly longer in either case for PS (P < 0.05). There is a correlation of the applied load with all other examined factors for PS and TB. Temperature and histological effects decrease for SB beyond 500 g. CONCLUSIONS: PS yields significantly higher temperatures and thermal tissue alterations on load levels higher than 500 g and is significantly slower for ISP compared to SB and TB. For ISP with PS, a maximum load of 400 g should be maintained.

The outcome after surgical therapy of bisphosphonate-associated osteonecrosis of the jaw--results of a clinical case series with an average follow-up of 20 months.

OBJECTIVES: The present case series evaluates the success rate of osteotomy and primary wound closure in patients with bisphosphonate-associated osteonecrosis of the jaw (BRONJ). MATERIALS AND METHODS: Eighty patients suffering from BRONJ were included in the study. All patients received intravenous bisphosphonate therapy and underwent osteotomy and primary wound closure according to a standardised protocol. After discharge, the patients were reviewed on a regular basis over an average time period of 20 months. RESULTS: During follow-up in 11 patients, a recurrence of BRONJ occurred in the former operation field. Seventeen patients died due to their underlying disease. The success rate of osteotomy and primary wound closure in the treatment of BRONJ was calculated at 84.2 % 20 months after surgery. The results showed non-significant difference concerning the outcome of surgery in the different clinical stages of BRONJ. CONCLUSIONS: In accordance with previous studies, stage-independent osteotomy and primary wound closure combined with antibiotics shall be deemed a viable treatment option in patients suffering from BRONJ. CLINICAL RELEVANCE: With a high success rate, osteotomy in combination with primary wound closure seems to be a viable alternative to more conservative protocols in the treatment of BRONJ.

The free scapular/parascapular flap as a reliable method of reconstruction in the head and neck region: a retrospective analysis of 130 reconstructions performed over a period of 5 years in a single department.

BACKGROUND: The scapular/parascapular free flap was described by Saijo in 1978 and has since then been widely used in reconstructive procedures. MATERIALS AND METHODS: This is a retrospective study, describing our experience with the use of free scapula/parascapular flap in 130 reconstructions over a period of 5 years in the Department of Oral and Maxillofacial Surgery of the University Hospital of Erlangen. Demographical data, data regarding the underlying pathology, flap raising details, microvascular anastomoses, early and late postoperative complications will be presented. RESULTS: The flap was raised without problems and the donor site was primarily closed in all cases. Sixteen flaps required revision. Five transplants were lost (failure rate of 3.85%). Loss of part of the flap was observed in 3 cases (2.3%). CONCLUSION: The free scapula/parascapular flap is a versatile and reliable flap that can find many applications in the reconstruction of complex head and neck defects.

Intraoral microvascular anastomosis for segmental mandibular reconstruction following removal of an ameloblastoma.

Cases of immediate bony microvascular reconstruction following segmental mandibulectomy in children are hard to find in the current literature. Moreover, microvascular segmental mandibular reconstruction that adopts an intraoral anastomosis technique has not been described so far. Therefore, the present clinical report aims at extending the armamentarium of bony microvascular reconstruction in pediatric cases of segmental mandibulectomy by highlighting an intraoral microvascular anastomosing technique.A 6-year-old boy, who suffered from an ameloblastoma of the mural type in the mandible, received a radical segmental mandibular resection because of the high recurrence rate of this tumor entity. Immediate reconstruction was carried out with a fibular double-barrel graft. Microvascular anastomoses were performed in an end-to-end fashion with the facial artery and vein as recipient vessels. The postoperative course was uneventful. There was no impairment of speech, deglutition, mastication, and facial nerve function. The facial appearance remained unobtrusive. On removal of the reconstruction plate 3 months after the reconstruction procedure, bleeding from the reconstructed mandibular segment indicated vascularization of the graft.It seems that segmental mandibulectomy and simultaneous microvascular bony reconstruction do not necessarily lead to impaired function as far as speech, deglutition, and mastication are concerned. Instead, the intraoral anastomosis technique allows waiving extraoral skin incisions and subsequent scarring, leaving the facial appearance unchanged and unobtrusive. Especially, the potential risk of stigmatization of the patient is avoided. Therefore, decision making in the choice of 1 or the other reconstruction option following segmental mandibulectomy should always consider the adoption of an intraoral anastomosing technique.

Enlargement of a Stafne cyst as an indication for surgical treatment - a case report.

Stafne cysts are often defined as static lesions located in the angle of the mandible. Consequently many authors have proposed that there is no need for surgical treatment of these bony defects on the lingual side of the mandible. This article describes the case of a 55-year-old patient, in whom a Stafne cyst showed a significant enlargement, reaching a size that necessitated surgical intervention because of the risk of pathological fracture. A literature search showed 5 additional similar cases, where progression in the size of a Stafne cyst could be radiographically documented. Consequently, the recommended management of these pseudocysts should be reconsidered.

Major histocompatibility complex class II polymorphisms are associated with the development of anti-resorptive agent-induced osteonecrosis of the jaw.

The aetiology of anti-resorptive agent-induced osteonecrosis of the jaw (ARONJ) is still under debate. Clinical and genetic risk factors are currently being investigated to help understand its pathogenesis. This case-control study analysed a large number of cancer patients (n = 230) under therapy with intravenous bisphosphonates, half of which were diagnosed with ARONJ. Multiple myeloma, greater patient age and the use of more than one bisphosphonate were identified as clinical risk factors on logistic regression analysis. In addition, 204 patients were genotyped for HLA-DRB1 and DQB1 and the allele frequencies were compared between ARONJ (n = 94) and unaffected cancer patients (n = 110). For the HLA class II alleles, a strong increase in the frequency of DRB1*15, DQB1*06:02, DRB1*01 and DQB1*05:01 was observed in the ARONJ group. These results were reinforced on analysis of the respective haplotypes, with DRB1*15-DQB1*06:02 being significantly associated with the development of ARONJ (odds ratio [OR] 2.5; 95% confidence interval [CI] 1.3-5.0). The presence of at least one of the haplotypes DRB1*15-DQB1*06:02 and DRB1*01-DQB1*05:01 was highly associated with the development of ARONJ (OR 3.0; 95% CI 1.7-5.5). The data in this study of a large number of cancer patients receiving intravenous bisphosphonates suggest that MHC class II polymorphisms represent genetic risk factors for the development of ARONJ. This result supports recent findings that inflammation and infection might play an important role in the pathogenesis of ARONJ.