Bortezomib-based induction therapies have shown to increase complete response rates and are used as an upfront therapy for newly diagnosed multiple myeloma patients. The standard treatment uses twice a week bortezomib at 1.3 mg/m^2 with dexamethasone PO on the day of and day after bortezomib, however, peripheral neuropathy is often a dose-limiting factor. For elderly patients with multiple co-morbidities and polypharmacy, we propose an alternate schedule of once a week bortezomib IV at 1.6 mg/m^2 with dexamethasone PO on the day of and day after bortezomib. In this phase II, open-labeled, multi-site study, we hypothesize that patients receiving weekly bortezomib will have comparable efficacy as the standard twice a week schedule with increased convenience and lower toxicity profile, especially related to peripheral neuropathy. METHODS: 50 patients with newly diagnosed symptomatic multiple myeloma who were ineligible for transplant or postponed transplant were enrolled from 12 Veterans Affairs hospitals. One cycle consisted of once a week 1.6 mg/m2 bortezomib IV (days 1, 8, 15, 22) plus dexamethasone PO on the day of and after bortezomib (days 1, 2, 8, 9, 15, 16, 22, 23) for 4 weeks, with the 5th week off of treatment. Responding patients could receive up to 6 cycles. RESULTS: The median age of patients was 71 ± 1.46 years (range: 50-89) with β-2 microglobulin of 5.80 ± 0.46 mg/L and c-reactive protein of 10.61 ± 5.54 mg/L. Patients also had multiple co-morbidities including cardiovascular disease (76%) renal insufficiency (54%) and pulmonary problems (36%) and were receiving a median of 13 concurrent medications at baseline. Of the fifty patients, 43 patients were evaluable for response. Seven patients received <1 cycle or died before response could be evaluated. An objective response rate of 79% was observed in 43 evaluable patients with 14% achieving nCR/CR, and at least VGPR in 44% of patients. The median progression-free survival was 9.6 months and overall survival was 46.5 months. The most common toxicity of all grades was thrombocytopenia (42%), lymphopenia (46%), asthenia (48%), and constipation (38%). Peripheral neuropathy occurred in 24% with grade 3 neuropathy occurring only in 6% of patients. In conclusion, a weekly bortezomib plus dexamethasone regimen is efficacious and safe, with lower neurotoxicity in elderly patients with newly diagnosed multiple myeloma complicated by extensive co-morbidities and polypharmacy.

Pain is a major personal and community burden throughout the world with currently limited treatment options for persistent pain due to unacceptable side effects, dependence or frank inefficacy. It is necessary to understand the anatomical and molecular pathways leading to pain to better cope with the current challenge of treating it.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成25年3月25日 水野紘樹氏の博士論文として提出された

No description available.

Platelets are anucleated cell fragments derived from megakaryocytes. They play a fundamental role in hemostasis, but there is rising evidence that they are also involved in immunological processes. Despite absence of a nucleus, human platelets are capable of de novo protein synthesis and contain a fully functional proteasome system, which is, in nucleated cells, involved in processes like cell cycle progression or apoptosis by its ability of protein degradation. The physiological significance of the proteasome system in human platelets is not yet fully understood and subject of ongoing research. Therefore, this study was conducted with the intention to outline the role of the proteasome system for functional characteristics of human platelets. For experimentation, citrated whole blood from healthy donors was obtained and preincubated with proteasome inhibitors. In addition to the commonly used bortezomib, the potent and selective proteasome inhibitor carfilzomib was selected as a second inhibitor to rule out agent-specific effects and to confirm that observed changes are related to proteasome inhibition. Irreversibly induced platelet activation and aggregation were not affected by proteasome blockade with bortezomib up to 24 hours. Conversely, proteasome inhibition led to enhanced threshold aggregation and agglutination up to 25 %, accompanied by partial alleviation of induced VASP phosphorylation of approximately 10-15 %. Expression of different receptors were almost unaffected. Instead, a significant increase of PP2A activity was observable in platelets after proteasome blockade, accompanied by facilitated platelet adhesion to coated surfaces in static experiments or flow chamber experiments. Carfilzomib, used for the first time in functional experimentation with human platelets in vitro, led to a dose-dependent decrease of proteasome activity with accumulation of poly ubiquitylated proteins. Like bortezomib, carfilzomib treatment resulted in enhanced threshold aggregation with attenuated VASP phosphorylation. As the main conclusion of this thesis, proteasome inhibition enhances the responsiveness of human platelets, provided by an alleviation of platelet inhibitory pathways and by an additional increase of PP2A activity, resulting in facilitated platelet adhesion under static and flow conditions. The proteasome system appears to be involved in the promotion of inhibitory counterregulation in platelets. The potential of proteasome inhibitors for triggering thromboembolic adverse events in patients must be clarified in further studies, in addition to their possible use for targeting platelet function to improve the hemostatic reactivity of platelets. / Thrombozyten sind kernlose Zellfragmente, welche aus Megakaryozyten gebildet werden. Sie spielen eine fundamentale Rolle in der Hämostase, aber es gibt immer mehr Hinweise, dass Thrombozyten auch in immunologischen Prozessen involviert sind. Trotz Fehlen eines Zellkerns haben humane Thrombozyten die Fähigkeit zur de novo Proteinsynthese und besitzen außerdem ein voll funktionstüchtiges Proteasomsystem, welches in kernhaltigen Zellen über den Proteinabbau an Prozessen wie dem Fortschreiten des Zellzyklus oder der Apoptose beteiligt ist. Die physiologische Bedeutung des Proteasomsystems in humanen Thrombozyten ist nicht vollständig geklärt und ist aus diesem Grund Gegenstand aktueller Forschung. Daher war es Ziel dieser Studie, die Rolle des Proteasomsystems für die funktionellen Eigenschaften humaner Thrombozyten zu erforschen. Für die Experimente wurde Citrat-Vollblut von gesunden Spendern gewonnen und mit Proteasom-Hemmstoffen vorinkubiert. Es wurde neben dem gängigen Bortezomib der potente und selektive Proteasom-Inhibitor Carfilzomib als zweiter Inhibitor eingesetzt, um substanzspezifische Effekte auszuschließen und zu bestätigen, dass die beobachteten Veränderungen auf der Proteasom-Inhibition beruhen. Die irreversibel induzierte Thrombozytenaktivierung und -aggregation wurde durch die Hemmung des Proteasoms mit Bortezomib bis zu 24 Stunden nicht beeinflusst. Allerdings führte die Proteasom-Hemmung zu einer verstärkten Schwellenwertaggregation und -agglutination um bis zu 25 % sowie zu einer partiellen Abschwächung der induzierten VASP-Phosphorylierung um etwa 10-15 %. Die Expression verschiedener Rezeptoren blieb nahezu unbeeinflusst. Stattdessen konnte unter Proteasom-Inhibition eine erhöhte Enzymaktivität der PP2A beobachtet werden, begleitet von einer erleichterten Thrombozytenadhäsion an beschichteten Oberflächen bei statischen Versuchen ober bei Flusskammerversuchen. Carfilzomib, welches erstmals für funktionelle Experimente mit menschlichen Thrombozyten in vitro eingesetzt wurde, führte zu einer dosisabhängigen Abnahme der Proteasom-Aktivität mit einer Akkumulation von poly ubiquitylierten Proteinen. Wie Bortezomib mündete die Behandlung mit Carfilzomib in einer verstärkten Schwellenwertaggregation und abgeschwächter VASP-Phosphorylierung. Die wichtigste Schlussfolgerung dieser Arbeit ist, dass die Inhibition des Proteasoms die Reaktivität humaner Thrombozyten erhöht, gekennzeichnet durch eine Abschwächung der hemmenden Signalwege der Thrombozyten und durch eine zusätzliche Erhöhung der PP2A-Enzymaktivität, was zu einer erleichterten Thrombozytenadhäsion unter statischen Verhältnissen und unter Flussbedingungen führt. Das Proteasomsystem scheint an der Förderung der hemmenden Gegenregulation in Thrombozyten beteiligt zu sein. Das Potenzial von Proteasom Inhibitoren, thromboembolische Nebenwirkungen bei Patienten auszulösen, muss in weiteren Studien geklärt werden, ebenso wie ihr möglicher Einsatz für die gezielte Beeinflussung der Thrombozytenfunktion zur Verbesserung der hämostatischen Reaktivität der Thrombozyten.

Bortezomib, ein selektiver und potenter Proteasominhibitor, wird experimentell in der Tumorzellforschung sowie therapeutisch in der Therapie des Multiplen Myeloms eingesetzt. Die Wirkung auf die Thrombozytenfunktion war bislang unzureichend untersucht. Daher evaluiert diese Studie die dosisabhängige Wirkung von Bortezomib auf die Viabilität, die Aggregation von gewaschenen Thrombozyten und auf aktivierende Signalwege in gewaschenen Thrombozyten. Die Thrombozytenviabilität war bei hohen Bortezomibkonzentrationen von 100 - 200 µM vermindert. Passend dazu verminderten 100 - 200 µM Bortezomib die Phosphorylierung der ERK1/2 und der Akt/PKB in humanen Thrombozyten. Im Gegensatz dazu hatten diese hohen Bortezomibkonzentrationen keinen Einfluss auf das Niveau der p38 MAP Kinase-Phosphorylierung in aktivierten Thrombozyten. Die Thrombozytenaggregation, induziert durch hohe Konzentrationen von Kollagen oder TRAP-6, blieb unter 0,1 nM - 200 µM Bortezomib unverändert. Zusammenfassend lässt sich sagen, dass Bortezomib weder die essenziellen, aktivierenden Signalwege noch die Initialisierung der Aggregation relevant beeinflusst. Das zeigt, dass diese Prozesse in Thrombozyten nicht abhängig von der Proteasomaktivität sind. Supramaximale Inhibierung des Proteasomsystems mit Bortezomibkonzentrationen von 100 µM oder mehr führen möglicherweise zu veränderter Thrombozytenreaktionsfähigkeit, welche unter Umständen durch unspezifische und potenziell toxische Effekte mit erniedrigter Zellviabilität verursacht werden. / Bortezomib, a selective and potent proteasome inhibitor, is used experimentally in tumor cell research and therapeutically in the treatment of multiple myeloma. Its effect on platelet function has been insufficiently studied. Therefore, this study evaluates the dose-dependent effects of bortezomib on viability, aggregation of washed platelets and activating signaling pathways in washed platelets. Platelet viability was tampered after incubation with high bortezomib concentrations of 100 - 200 µM. Fittingly, 100 - 200 µM bortezomib decreased phosphorylation of ERK1/2 and Akt/PKB in human platelets. In contrast, these high concentrations of bortezomib had no effect on the level of p38 MAP kinase phosphorylation in activated platelets. Furthermore platelet aggregation induced by high concentrations of collagen or TRAP-6 remained unchanged under the influence 0.1 nM - 200 µM bortezomib. In conclusion, bortezomib does not relevantly affect essential activating signaling pathways or the initialization of aggregation. This indicates that these processes in platelets are not dependent on proteasome activity. Supramaximal inhibition of the proteasome system with bortezomib concentrations of 100 µM or above may lead to altered platelet responsiveness, which may be accompanied by nonspecific and potentially toxic effects associated with decreasing cell viability.

Ziel dieser Arbeit war die Analyse der Pilotdaten eines Kollektivs, bestehend aus 25 Patient*innen (medianes Alter 66 Jahre) mit MM unter BTZ-Therapie, um eine bessere Durchführbarkeit der gesamten „Bortezomib induzierten schmerzhafte Neuropathie: Risikofaktoren, Resilienz und Resolution“ Studie zu ermöglichen. Dazu sollten unter anderem die Charakteristika bzw. die Repräsentativität der Studienpopulation und die genaue Ausprägung der BIPN, sowie etwaige Risikofaktoren untersucht werden. Um den Ausprägungsgrad der Neuropathie abschätzen zu können wurden die Kriterien des mTCNS erhoben. Zur näheren Beurteilung der Beteiligung unterschiedlicher Nervenfaserklassen wurden bei den Patient*innen eine Elektroneurographie, eine QST, eine Hautbiopsie und eine C-Faser-Messung durchgeführt. Durch eine ausführliche Anamnese und Verteilung von Fragebögen (BPI, NPSI, BDI, STAI-G, SF-12) wurde eine genaue Beschreibung des Kollektivs ermöglicht. Im Zuge der BDNF-Bestimmung wurden zwei Verfahren (ELLA-Gerät und ELISA) miteinander verglichen. 80 % der Patient*innen wiesen nach Maßgabe des mTCNS eine PNP auf, die in der Elektroneurographie als überwiegend axonale, sensibel betonte sensomotorische PNP klassifiziert wurde. Die QST zeigte eine vorwiegende Beteiligung von Aδ- und C-Fasern, wohingegen in der Hautbiopsie nur eine leichte Reduktion der IENFD zu sehen war. Bei der BDNF-Bestimmung wurde eine starke Korrelation zwischen den Messreihen mittels ELISA und ELLA-Gerät detektiert und nur geringfügige Hinweise für eine Assoziation des BDNF-Serumspiegel mit dem Schwergrad der Neuropathie gefunden. Als mögliche Risikofaktoren für die Entwicklung einer BIPN wurden das Alter, die Dauer des Nikotinabusus, der BMI und eine vorbestehende Neuropathie ermittelt. Für die Nachfolgestudie ergibt sich hieraus, dass unser Studienkollektiv gut mit Populationen in der Literatur vergleichbar ist. Korrekturen im Rekrutierungsprozess sind notwendig, um vermehrt Patient*innen mit Neudiagnose einschließen zu können. Durch Veränderung des Studienablaufs soll die Akzeptanz bei den Patient*innen erhöht werden. Bei der BIPN sind sowohl größere als auch kleinkalibrige Fasern mitbetroffen, wobei sich letzteres vor allem in Form einer funktionellen Beeinträchtigung in der QST demaskiert. Eine Etablierung von spezifischen Normwerten für die BDNF-Bestimmung mittels ELLA-Gerät sollte realisiert und die gefundenen möglichen Risikofaktoren für die Entstehung einer Neuropathie im prospektiven Setting weiter untersucht werden. / The aim of this work was to analyse the pilot data of a collective consisting of 25 patients (median age 66 years) with MM undergoing BTZ therapy in order to improve the feasibility of the entire ‘Bortezomib-induced painful neuropathy: risk factors, resilience and resolution’ study. Among other things, the characteristics and representativeness of the study population and the exact severity of BIPN, as well as any risk factors, should be investigated. In order to be able to estimate the degree of neuropathy, the criteria of the mTCNS were determined. Electroneurography, a QST, a skin biopsy and a C-fibre measurement were carried out on the patients to assess the involvement of different nerve fibre classes in more detail. A detailed medical history and distribution of questionnaires (BPI, NPSI, BDI, STAI-G, SF-12) enabled a precise description of the collective. Two methods (ELLA device and ELISA) were compared with each other in the course of BDNF determination. According to the mTCNS, 80% of the patients had PNP, which was classified as predominantly axonal, sensory-emphasised sensorimotor PNP on electroneurography. The QST showed predominant involvement of Aδ and C fibres, whereas the skin biopsy showed only a slight reduction in IENFD. In the BDNF determination, a strong correlation was detected between the series of measurements using ELISA and ELLA device and only slight indications of an association of the BDNF serum level with the severity of the neuropathy were found. Age, duration of nicotine abuse, BMI and pre-existing neuropathy were identified as possible risk factors for the development of BIPN. For the follow-up study, this means that our study population is well comparable with populations in the literature. Corrections in the recruitment process are necessary in order to be able to include more patients with a new diagnosis. Changes to the study procedure should increase patient acceptance. BIPN affects both large and small calibre fibres, with the latter manifesting itself primarily in the form of functional impairment in the QST. The establishment of specific standard values for BDNF determination using the ELLA device should be realised and the possible risk factors for the development of neuropathy identified should be further investigated in a prospective setting.

A distrofia muscular progressiva no cão Golden Retriever (GRMD) é uma miopatia genética homóloga à Distrofia Muscular de Duchenne (DMD) que acomete humanos. A utilização do modelo canino é considerada ideal uma vez que a progressão da doença, as manifestações clínicas e a massa corporal se assemelham às apresentadas por meninos doentes. Sabe-se que a degradação de proteínas musculares e atrofia muscular estão intimamente relacionadas com a atuação do sistema catalítico ubiquitina-proteassoma. Estudos utilizando camundongos com distrofia muscular e músculos de humanos DMD (in vitro) tratados com inibidores de proteassoma (MG-132 e bortezomibe) apresentaram restauração da distrofina e das proteínas associadas à distrofina e melhora do fenótipo histopatológico. Nesta pesquisa, foram avaliados cinco cães GRMD, dois tratados com Bortezomibe e três controles. Coletou-se biopsia muscular antes de iniciar o tratamento e ao término do mesmo, para os testes de histologia, ultraestrutura e imunohistoquímica. Foram realizados semanalmente testes bioquímicos e em cada ciclo de aplicação do bortezomibe foi mensurada a taxa de inibição do proteassoma. Na primeira biopsia todos os cães apresentaram morfologia histopatológica das fibras musculares semelhantes. Ao final do tratamento, os cães tratados apresentaram menor deposição de tecido conjuntivo e infiltração de células inflamatórias que os cães não tratados julgados por meio da histologia, morfometria do colágeno e ultraestrutura. Na ultraestrutura observamos infiltração de macrófagos nas fibras, material degenerado, fibroblastos ativados e deposição e tecido conjuntivo em endomísio e perimísio das fibras. Pela análise de imunohistoquímica, não constatamos presença de distrofina na membrana sarcoplasmática de ambos os grupos avaliados. Entretanto, os cães tratados apresentaram maior expressão das proteínas &alpha; e &beta; distroglicano, o que sugere uma melhora no fenótipo histopatológico da doença. Nos cães não tratados observou-se maior expressão de phospho- NF&kappa;B e TGF-&beta;1 sugerindo maior ativação de fatores pró-apoptóticos e moléculas inflamatórias, e maior deposição de tecido conjuntivo, respectivamente. Os testes de inibição do proteassoma indicaram maior inibição nas células sanguíneas uma hora após a aplicação do bortezomibe e estes de comportaram de forma dose-dependente. Concluímos que os inibidores de proteassoma podem melhorar a morfologia das fibras musculares dos cães GRMD tratados, diminuindo a deposição de colágeno e infiltração de células inflamatórias, bem como restaurar parte das proteínas associadas à distrofina na membrana sarcoplasmática das fibras musculares. / The Golden Retriever Muscular Dystrophy (GRMD) is a genetic myopathy homologue to Duchenne Muscular Dystrophy (DMD) in human. Use of canine model is the better because the disease development, clinical signs and body mass are closed to the sick children. It has been known muscle protein proteolysis and muscle atrophy are related with ubiquitin-proteasome pathway. Researches using mdx mice and human muscle (in vitro) treated with proteasome inhibitor (MG-132 and bortezomib) showing rescue of the dystrophin and associated proteins and improvement of histopatologycal phenotype. In this research, were analyzed five GRMD dogs, two dogs were treated with Bortezomib and three dogs were control. Muscle biopsies were collected before the treatment and after the treatment to histology, ultrastructure and immunohistochemical muscle analysis. Biochemistry analyses were made once a week and measurement of proteasome inhibition was analysed in each cycle of Bortezomib administration. In the first byopsy, all the dogs showing closed histophatological morphology of muscle fibers. At the end of treatment, the treated dogs had lower connective tissue deposition and inflammatory cells infiltration than untreated dogs by histology, collagen morphometry and ultrastructural analysis. We noted by ultrastructural analysis macrophages inside the fibers, degenerated products, activated fibroblasts and connective tissue deposition in edomisium and perimisium of the fibers. The dystrophin immunohistochemistry was not presence in sarcoplasmatic membrane in both groups. However, the treated dogs showing bigger expression of &alpha; and &beta;-dystroglycan, this fact means improved of disease histopathology phenotype. The untreated dogs had bigger expression of phospho-NF&kappa;B and TGF-&beta;1, suggesting bigger activation of pro-apoptotic factors, inflammatory molecules and bigger connective tissue deposition respectively. The proteasome inhibition tests indicated bigger inhibition in the blood cells one hour post doses of bortezomib and was dose-dependent pathway. In conclusion, the proteasome inhibitors may improve the appearance of GRMD muscle fibers, lowered the connective tissue deposition and infiltration of inflammatory cells, likewise to rescue the dystrophin- associated proteins in the muscle fiber membrane.

A distrofia muscular progressiva no cão Golden Retriever (GRMD) é uma miopatia genética homóloga à Distrofia Muscular de Duchenne (DMD) que acomete humanos. A utilização do modelo canino é considerada ideal uma vez que a progressão da doença, as manifestações clínicas e a massa corporal se assemelham às apresentadas por meninos doentes. Sabe-se que a degradação de proteínas musculares e atrofia muscular estão intimamente relacionadas com a atuação do sistema catalítico ubiquitina-proteassoma. Estudos utilizando camundongos com distrofia muscular e músculos de humanos DMD (in vitro) tratados com inibidores de proteassoma (MG-132 e bortezomibe) apresentaram restauração da distrofina e das proteínas associadas à distrofina e melhora do fenótipo histopatológico. Nesta pesquisa, foram avaliados cinco cães GRMD, dois tratados com Bortezomibe e três controles. Coletou-se biopsia muscular antes de iniciar o tratamento e ao término do mesmo, para os testes de histologia, ultraestrutura e imunohistoquímica. Foram realizados semanalmente testes bioquímicos e em cada ciclo de aplicação do bortezomibe foi mensurada a taxa de inibição do proteassoma. Na primeira biopsia todos os cães apresentaram morfologia histopatológica das fibras musculares semelhantes. Ao final do tratamento, os cães tratados apresentaram menor deposição de tecido conjuntivo e infiltração de células inflamatórias que os cães não tratados julgados por meio da histologia, morfometria do colágeno e ultraestrutura. Na ultraestrutura observamos infiltração de macrófagos nas fibras, material degenerado, fibroblastos ativados e deposição e tecido conjuntivo em endomísio e perimísio das fibras. Pela análise de imunohistoquímica, não constatamos presença de distrofina na membrana sarcoplasmática de ambos os grupos avaliados. Entretanto, os cães tratados apresentaram maior expressão das proteínas &alpha; e &beta; distroglicano, o que sugere uma melhora no fenótipo histopatológico da doença. Nos cães não tratados observou-se maior expressão de phospho- NF&kappa;B e TGF-&beta;1 sugerindo maior ativação de fatores pró-apoptóticos e moléculas inflamatórias, e maior deposição de tecido conjuntivo, respectivamente. Os testes de inibição do proteassoma indicaram maior inibição nas células sanguíneas uma hora após a aplicação do bortezomibe e estes de comportaram de forma dose-dependente. Concluímos que os inibidores de proteassoma podem melhorar a morfologia das fibras musculares dos cães GRMD tratados, diminuindo a deposição de colágeno e infiltração de células inflamatórias, bem como restaurar parte das proteínas associadas à distrofina na membrana sarcoplasmática das fibras musculares. / The Golden Retriever Muscular Dystrophy (GRMD) is a genetic myopathy homologue to Duchenne Muscular Dystrophy (DMD) in human. Use of canine model is the better because the disease development, clinical signs and body mass are closed to the sick children. It has been known muscle protein proteolysis and muscle atrophy are related with ubiquitin-proteasome pathway. Researches using mdx mice and human muscle (in vitro) treated with proteasome inhibitor (MG-132 and bortezomib) showing rescue of the dystrophin and associated proteins and improvement of histopatologycal phenotype. In this research, were analyzed five GRMD dogs, two dogs were treated with Bortezomib and three dogs were control. Muscle biopsies were collected before the treatment and after the treatment to histology, ultrastructure and immunohistochemical muscle analysis. Biochemistry analyses were made once a week and measurement of proteasome inhibition was analysed in each cycle of Bortezomib administration. In the first byopsy, all the dogs showing closed histophatological morphology of muscle fibers. At the end of treatment, the treated dogs had lower connective tissue deposition and inflammatory cells infiltration than untreated dogs by histology, collagen morphometry and ultrastructural analysis. We noted by ultrastructural analysis macrophages inside the fibers, degenerated products, activated fibroblasts and connective tissue deposition in edomisium and perimisium of the fibers. The dystrophin immunohistochemistry was not presence in sarcoplasmatic membrane in both groups. However, the treated dogs showing bigger expression of &alpha; and &beta;-dystroglycan, this fact means improved of disease histopathology phenotype. The untreated dogs had bigger expression of phospho-NF&kappa;B and TGF-&beta;1, suggesting bigger activation of pro-apoptotic factors, inflammatory molecules and bigger connective tissue deposition respectively. The proteasome inhibition tests indicated bigger inhibition in the blood cells one hour post doses of bortezomib and was dose-dependent pathway. In conclusion, the proteasome inhibitors may improve the appearance of GRMD muscle fibers, lowered the connective tissue deposition and infiltration of inflammatory cells, likewise to rescue the dystrophin- associated proteins in the muscle fiber membrane.

The number of known monogenic rare diseases (~7000) exceeds the number of effective treatments (~500) by more than an order of magnitude underlining the pressing need for generalizable therapeutic approaches for this class of conditions. In this regard, the majority of recessive and x-linked recessive disorders are caused by missense mutations encoding proteins that frequently have residual function but are rapidly degraded by the 26S proteasome. Bortezomib is a small molecule that inhibits the 26S proteasome and has been approved for use in patients for an unrelated condition; multiple myeloma. Previous work has shown that, for a small number of disorders, bortezomib can inhibit the degradation of the mutant protein, thereby increasing the protein level and activity, holding out the promise of a beneficial therapeutic effect by the repurposing of this agent. We present here a high level western blot based survey of nine recessive disorders to characterize the general effectiveness of such an approach. Thirteen patient fibroblast cell lines comprising 9 different diseases with 19 known mutations were selected on the basis of missense mutations protein expression data when available. The cell lines were incubated with bortezomib (10 nM and 50 nM; 24 hrs) and levels of the mutated protein were quantified by western blot. Unfortunately, no consistent, appreciable increase was observed for any of the conditions tested. The general therapeutic value of re-purposing bortezomib for recessive and x-linked diseases appears limited at best. The few reported cases of bortezomib successfully working in increasing mutated protein levels appear to be the exceptions and not the norm. Moreover successes are more often limited to cell lines carrying a transgene expressing the mutated protein rather than endogenous mutated protein in patient cell lines.