Several wild-type poxviruses have emerged as potential oncolytic viruses (OVs), including orf virus (OrfV), and vaccinia virus (VV). Oncolytic VVs have been modified to include attenuating mutations that enhance their tumour selective nature, but these mutations also reduce overall viral fitness in cancer cells. Previous studies have shown that a VV (Western Reserve) with its E3L gene replaced with the E3L homologue from, OrfV (designated VV-E3LOrfV), maintained its ability to infect cells in vitro, but was attenuated compared to its parental VV in vivo. Our goal was to determine the safety and oncolytic potential VV-E3LOrfV, compared to wild type VV and other attenuated recombinants. VV-E3LOrfV, was unable to replicate to the same titers and was sensitive to IFN compared to its parental virus and other attenuated VVs in normal human fibroblast cells. The virus was also less pathogenic when administered in vivo. Viral replication, spread and cell killing, as measures of oncolytic potential in vitro, along with in vivo efficacy, were also observed.. The Parapoxvirus, OrfV has been shown to have a unique immune-stimulation profile, inducing a number of pro-inflammatory cytokines, as well as potently recruiting and activating a number of immune cells. Despite this unique profile, OrfV is limited in its ability to replicate and spread in human cancer cells. Various strategies were employed to enhance the oncolytic efficacy of wild-type OrfV. A transient transfection/infection screen was created to determine if any of the VV host-range genes (C7L, K1L, E3L or K3L) would augment OrfV oncolysis. Combination therapy, including the use of microtubule targeting agents, Viral Sensitizer (VSe) compounds and the addition of soluble VV B18R gene product were employed to see if they also enhance OrfV efficacy. Unfortunately, none of the strategies mentioned were able to enhance OrfV.

Nowell (1976) first proposed that unless cytotoxic cancer therapy eradicates all tumor cells, genetic or heritable variation within heterogeneous tumors will inevitably lead to the evolution of chemotherapeutic resistance through clonal selection. This evolutionary hypothesis was formalized by Goldie and Coldman (1979), who developed one of the earliest mathematical kinetic models of resistance evolution in neoplasms. Their model predicted that the likelihood of response and cure would be increased in combination vs single agent cytotoxic therapies. In a later study, Gardner (2002) developed a computational kinetic model to predict chemotherapeutic combinations, doses, and schedules most likely to result in patient response and prolonged life. This model predicts that combination therapy involving both cytotoxic and cytostatic drugs will be more effective than combination therapy involving only cytotoxic drugs. Thus far, no systematic evaluation of the Goldie and Coldman and Gardner hypotheses have been conducted in the metastatic clinical trial setting. Here I test these hypotheses using the results of over 700 phase II, III and II/III clinical trials. I show that, as predicted by Goldie and Coldman, both overall response rate and overall survival were greater in combination arms. Moreover, median duration of response – the key indicator of the rate of resistance evolution - was also greater in combination vs single agent arms. These results suggest that generally combination chemotherapy is more effective than single agent therapy for advanced solid tumors as predicted by Goldie and Coldman (1979) hypothesis and that, at least in the metastatic setting, the potential disadvantages of combination therapy with respect to accelerated resistance evolution are outweighed by the greater waiting times for resistance mutations to arise. By contrast, although combination cytotoxic and cytostatic therapy is associated with a greater average overall response rate than multi agent cytotoxic therapy, this is not the case for both median duration of response and overall survival. Hence, there is no evidence that, in contrast to the predictions of the Gardner (2002) model, combination cytotoxic and cytostatic therapy decreases the rate of resistance evolution relative to that obtaining under combination cytotoxic therapy.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22694号 / 医博第4638号 / 新制||医||1045(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 竹内 理, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Mu-opioid receptor (MOR) agonists are effective agents for pain management, but are also limited by a number of undesirable effects. One approach to enhance the therapeutic effects and minimize the undesirable effects of MOR agonists may be to combine MOR agonists with an adjunct targeting a different receptor system. This targeted medical approach, known as “combination therapy”, aims to augment the desired effects of the MOR agonist (i.e. antinociception) and/or diminish the undesirable deleterious side effects of the MOR agonist. This dissertation investigated the utility of this approach in an assay of thermal nociception and schedule-controlled responding in male rhesus monkeys with three aims. One aim determined the utility of N-methyl D-aspartate (NMDA) receptor antagonists to selectively enhance MOR agonist antinociception. A second identified the pharmacological determinants of antinociceptive interactions between a nociceptin opioid peptide (NOP) receptor agonist and MOR agonists. A third aim investigated the potential for fixed-proportion mixtures of a competitive MOR antagonist and MOR agonist to manipulate antinociceptive efficacy. Experimental results did not support the utility of NMDA antagonists as adjuncts to selectively enhance MOR agonist antinociception. Furthermore, the antinociceptive interactions between a NOP agonist and MOR agonists were modest and occurred under a narrow range of conditions. Finally, fixed proportion MOR antagonist-agonist mixtures were effective in manipulating antinociceptive in vivo efficacy. In conclusion, this dissertation does not provide strong empirical evidence that a combination therapy approach will result in clinically effective and selective enhancement of MOR agonist analgesia. The dissertation concludes with proposed strategies and novel preclinical methods to enhance preclinical-to-clinical translation of effective candidate analgesics.

Bakgrund: Crohns sjukdom är en form av inflammatorisk tarmsjukdom som kännetecknas av kronisk histologisk inflammation, en autoimmun sjukdom som inte är medicinskt botbar men det finns olika medicinska terapier som kan kontrollera symtomen. Det förekommer omväxlande perioder med skov och lugn, operation behövs ibland, medicineringen är konstant och alla dessa faktorer leder till försämrad livskvalitet. Crohns sjukdom skadar ofta ileum men det kan påverka vilket segment av mag-tarmkanalen som helst från mun till perianalt område, lesionerna kan ha formen av fläckar, med vissa delar påverkade medan andra förblir helt normala. Incidensen av Crohns sjukdom i Sverige är mellan 5 och 10 per 100 000 och prevalensen är cirka 200 per 100 000. Medan dödsfallet med Crohns sjukdom minskar, ökar de andra problemen som är förknippade med Crohns sjukdom och detta sätter press på sjukvården. För att hålla sjukdomen under kontroll behöver patienter farmakologisk induktionsbehandling i början av sjukdomen och underhållsbehandling under resten av livet. Just för att behandlingen behövs på lång sikt (livet ut) har det varit absolut nödvändigt att hitta den bästa behandlingsstrategin. Målet med behandlingen är att göra patienten symptomfri med hjälp av medicinering. Mål: Syftet med detta examensarbete var att ta reda på hur tillgängliga biologiska läkemedel ska användas för att få optimal effekt, närmare bestämt om de biologiska läkemedlen ska användas som monoterapi eller i kombination med andra läkemedel vid behandling av Crohns sjukdom. Metod: PubMed-databasen har använts för att söka, välja och analysera fem relevanta randomiserade kontrollerade kliniska prövningar om Crohns sjukdom. Nyckelordet som användes för att hitta alla dessa fem relevanta artiklar var ''Crohn's combination therapy''. Resultat: Resultaten i den första artikeln påpekar att kombinationsbehandling med infliximab + azatioprin hade bättre resultat jämfört med infliximab monoterapi och infliximab monoterapi hade bättre resultat jämfört med azatioprin monoterapi för att uppnå klinisk remission och slemhinneläkning. Den andra artikeln strider mot den första studien och säger att om tillräckliga läkemedelskoncentrationer uppnås och bibehålls genom terapeutisk läkemedelsövervakning med infliximab, kanske kombinationsterapi med tiopuriner inte behövs för att uppnå önskade kliniska resultat. Den tredje artikeln drog slutsatsen att adalimumab var överlägsen tiopuriner i att förhindra endoskopiskt återfall av Crohns sjukdom hos patienter med hög risk för återfall efter operation. Den fjärde artikeln identifierade ingen effektivitetsfördel med kombinationsbehandling med adalimumab + immunmodulatorer jämfört med adalimumab monoterapi. Den femte artikeln drog slutsatsen att vedolizumab i kombination med stabila doser av kortikosteroider inducerade mer effektivt klinisk remission än antingen placebo plus kortikosteroider eller enbart vedolizumab. Slutsatser: En universell slutsats kan inte dras om fördelen med kombinationsbehandling bestående av biologiska och immunsuppressiva läkemedel jämfört med biologisk monoterapi. Olika tillvägagångssätt är nödvändiga för varje enskild patient eftersom medicinen kan vara olika för induktions- och underhållsterapier, sjukdomsvaraktigheten påverkar hur patienten kommer att reagera på mediciner och medicineringen som Crohns sjukdoms-patienter fick tidigare kan också påverka hur patienten kommer att svara på medicinering. / Background: Crohn's disease is a form of inflammatory bowel disease characterized by chronic histological inflammation, an autoimmune disorder that is not medically curable but there are various medical therapies able to control the symptoms. It has periods of exacerbation and calmness, with surgery needed sometimes, the use of medication is constant and all these factors lead to an impaired quality of life. Crohn's disease often damages the ileum but it can affect any segment of the gastrointestinal tract from mouth to perianal area, the lesions may take the form of patches, with some sections affected while others remain perfectly normal. The incidence of Crohn's disease in Sweden is between 5 and 10 per 100,000 and the prevalence is around 200 per 100,000. While the fatal burden is declining, the non-fatal burden is increasing. In order to keep the disease under control, patients require pharmacological induction treatment at the beginning and maintenance treatment for the rest of their life. Because the treatment is needed long-term, finding the best treatment strategy has become imperative. The goal with the therapy is to make the patient symptom-free with the help of medication. Objective: The purpose of this degree project is to find out how biological drugs should be used in order to have optimal effect, more specifically, if the biological medicines should be used as monotherapy or in combination with other medicines in the treatment of Crohn's disease. Method: PubMed database has been used to search, select and analyze five relevant randomized controlled clinical trials about Crohn’s disease. The keyword used to find all these five relevant articles were ''Crohn's combination therapy''. Results: The results in the first article point out that Infliximab + Azathioprine combination therapy had better results compared to infliximab monotherapy and infliximab monotherapy had better results compared with Azathioprine monotherapy in achieving clinical remission and mucosal healing. The second article contradicts the first study and states that if adequate drug concentrations are achieved and maintained through therapeutic drug monitoring with infliximab, combination therapy with thiopurines may not be required to achieve desired clinical results. The third article concluded that adalimumab was superior to thiopurines in preventing endoscopic recurrence of Crohn's disease in patients with high risk of recurrence after surgery. The fourth article identified no efficacy benefit of adalimumab + immunomodulators combination therapy compared to adalimumab monotherapy. The fifth article concluded that vedolizumab in combination with stable doses of corticosteroids induced more efficient clinical remission than either placebo plus corticosteroids or vedolizumab alone. Conclusions: A universal conclusion cannot be drawn regarding the superiority of combination treatment consisting of biological and immunosuppressive medicines compared to biological monotherapy. Different approaches are necessary for every individual patient because the medication can be different for induction and maintenance therapies, the duration of the disease affects how the patient will respond to medication, and the medication that the Crohn’s disease patient took in the past may also affect how the patient will respond to medication.

DNA-damaging chemotherapeutics are a consistently employed for the treatment of cancer, and are regularly part of first-line combination therapeutic regimens. However, these regimens often display limited activity in cancers including of the lung and breast. Novel therapeutic strategies are urgently required. Understanding the molecular mechanisms regulating DNA-damaging chemotherapeutics activity will lead to such novel strategies. Activating transcription factor 3 (ATF3) is a stress inducible gene that plays a significant role in regulating cellular stress including DNA damage. This thesis investigates the role of ATF3 in mediating the cytotoxic effects of two commonly employed DNA-damaging chemotherapeutics, cisplatin and doxorubicin. This study also identifies other independent ATF3 inducing agents in potential novel combination therapeutic strategies. In this study, cell line models of cisplatin-resistance were generated independently from two non-small cell lung cancer (NSCLC) cell lines, Calu6 and H23. Full transcriptome RNA-sequencing analysis identified ATF3 as the most highly dysregulated apoptosis regulatory gene in the cisplatin-resistant compared to their respective parental cell lines following treatment with cisplatin. Further characterization identified cisplatin induced activation of JNK as the key regulator of ATF3 induction in these cell lines. Restoring JNK activity resulted in induced ATF3 expression and re-sensitization of the resistant cell lines to cisplatin treatment. FDA-approved 1200 compound library screens were employed to identify agents that can enhance cisplatin cytotoxicity as well as whose cytotoxicity was dependent on ATF3 expression. Vorinostat, an HDAC inhibitor, was identified in both screens and importantly displayed synergistic cytotoxicity in combination with cisplatin. In addition, ATF3 was also induced with treatments of the DNA damaging agent doxorubicin in these NSCLC cell lines including in the cisplatin resistant models. This work suggested a different mechanism of ATF3 induction by doxorubicin and the potential role of ATF3 in mediating doxorubicin cytotoxicity was further investigated in breast cancer cells. Doxorubicin robustly increased ATF3 expression in human breast cancer cell lines and tumor tissue ex-vivo. Loss of ATF3 in MEFs resulted in reduced sensitivity to doxorubicin treatment compared to wild-type MEFs. Employing the same library screen as above, compounds with ATF3 dependent mechanisms that could enhance doxorubicin cytotoxicity were identified. Vorinostat, as well as, the nucleoside analogues trifluridine and 6-mercaptopurine induced ATF3 expression and enhanced doxorubicin cytotoxicity. This study demonstrated that ATF3 plays an important role in mediating the cytotoxic effect of the DNA-damaging chemotherapeutics cisplatin and doxorubicin. It also provides rationale for ATF3 as a potential therapeutic target, and for the incorporation of ATF3 inducing agents in novel combination therapeutic strategies.

Magister Scientiae Dentium - MSc(Dent) / Candida glabrata (C. glabrata/ Cg) is a pathogenic organism that is increasingly developing frank innate and acquired resistance to the most commonly used antifungal agents, namely, azole group of antifungals. Furthermore, C. glabrata-associated oropharyngeal infections affecting immunocompromised patients, are more difficult to treat and the development of resistance worsen the prognosis. Molecular studies related the emergence of resistance in C. glabrata to the upregulation of ATP-binding cassette (ABC) transporter genes, which work by reducing drug concentration within the cell via drug efflux mechanism, and among these genes, CgCDR1 is considered to play a major role in resistance development. Thus, in order to overcome this problem, several combinations of antifungal agents are being studied. Aim: To evaluate the effect of a combination therapy of fluconazole and amphotericin B on the growth and CDR1 gene expression of C. glabrata. Research design and methodology: This in-vitro study evaluated the effect of a combination therapy of fluconazole and amphotericin B on the growth of C. glabrata and related it to the expression of CgCDR1 resistance gene. C. glabrata was revived in brain heart infusion (BHI) broth and later inoculated onto agar plates. Following overnight incubation, 5 colonies were transferred using a sterile loop into 2 ml of phosphate buffered saline (PBS) solution to establish McFarland (Mcf) standard. Later, the solution was diluted by transferring 200 μL to 400 ml of yeast peptone dextrose (YPD) agar (flask 1). From (flask 1), 90 ml, 99 ml and 89 ml of inoculum were allocated into 3 separate flasks, into which 10 ml fluconazole, 1 ml amphotericin B and 11 ml combination (10 ml fluconazole + 1 ml amphotericin B) were added, respectively. The inoculums were left to settle for 20 minutes, then incubated at 37oC with serial dilutions carried after 30 minutes, 2, 4, 6 and 24 hours. From the 96-microtiter plate, 10 μL for each treatment arm and time interval were transferred from selected wells and onto 30 Casein-peptone Soymeal-peptone (CASO) agar plates, and incubated for 24 hours. After incubation, the number of colonies were counted using an automated colony counter, to establish colony forming unit (CFU)/ml. CgCDR1 gene expression was analyzed using real time polymerase chain reaction. After 6 hours of incubation, a sample was taken from each treatment arm, transferred into CASO agar plates and incubated for 24 hours at 37oC. After establishing Mcf, gene extraction and gene expression were carried out according to manufacturer’s instructions. Results and discussion: No significant difference between the effect of the combination and amphotericin B was evident regarding C. glabrata growth. However, the combination therapy was more effective against C. glabrata than fluconazole, with a marked decrease in candidal growth at 30 minutes and 6 hours. Furthermore, the expression of CgCDR1 gene at 6 hours contact time was more pronounced in the samples of C. glabrata treated with the combination therapy, compared to that of the monotherapy. Conclusion: The combination therapy had better effect on the growth of C. glabrata than fluconazole monotherapy. On the other hand, the expression of CgCDR1 was detected in the samples of C. glabrata treated with the combination therapy, suggesting the ability of the yeast to adapt and develop resistance in such environment.

C-reactive protein (CRP) is a component of innate immunity. The concentration of CRP in serum increases in microbial infections including Streptococcus pneumoniae infection. Employing a mouse model of pneumococcal infection, it has been shown that passively administered human wild-type CRP protects mice against infection, provided that CRP is injected into mice within two hours of administering pneumococci. Engineered CRP (E-CRP) molecules have been reported recently; unlike wild-type CRP, passively administered E-CRP protected mice against infection even when E-CRP was injected into mice after twelve hours of administering pneumococci. The current study was aimed at comparing the protective capacity of E-CRP with that of an antibiotic clarithromycin. We established a mouse model of pneumococcal infection in which both E-CRP and clarithromycin, when used alone, provided minimal but equal protection against infection. In this model, the combination of E-CRP and clarithromycin drastically reduced bacteremia and increased survival of mice when compared to the protective effects of either E-CRP or clarithromycin alone. E-CRP was more effective in reducing bacteremia in mice treated with clarithromycin than in untreated mice. Also, there was 90% reduction in antibiotic dosing by including E-CRP in the antibiotic-treatment for maximal protection of infected mice. These findings provide an example of cooperation between the innate immune system and molecules that prevent multiplication of bacteria, and that should be exploited to develop novel combination therapies for infections against multidrug-resistant pneumococci. The reduction in antibiotic dosing by including E-CRP in the combination therapy might also resolve the problem of developing antibiotic resistance.

Decreasing the impact of stressors on the body remains an important area of study for the affected population. While there is evidence showing that cognitive behavior therapy (CBT), a psychotherapy approach, results in decreased stress, little was found about the effects of chiropractic treatment (CC) on stress. The purpose of this quantitative archival study was to determine whether the combination therapy of CC and CBT was more effective in decreasing stress than CBT independently. Cognitive neuropsychology served as the theoretical lens. Client data from a mental health and chiropractic care center on the West coast (N = 112) were divided into 2 treatment groups, CBT and CC and CBT alone. Pre and posttreatment data were collected on stress, anxiety, and nerve conduction. ANOVA test results indicated that there were no statistically significant differences in the mean change scores between the 2 groups in terms of individual participants' stress, anxiety, and nerve interference. Although there was no significant interaction effect, results showed that both the combination therapy and CBT alone led to a decrease in stress and anxiety and an increase in the nerve conduction of participant's posttreatment. While this archival study did not yield evidence of the benefits of CC for stress-related disorders, its results suggest that future researchers should pursue more direct efforts to evaluate the effects of combination therapies. Considering the high number of people who experience stress-related challenges, the incorporation of CC along with a psychological treatment might engender positive social change for individuals and healthcare practitioners through the potential reduction of stress.

Indiana University-Purdue University Indianapolis (IUPUI) / Pancreatic ductal adenocarcinoma (PDAC) is an extremely deadly disease characterized by aggressive metastasis and therapeutic resistance. Reactive stroma in pancreatic tumors contributes to tumor signaling, fibrosis, inflammation, and hypoxia. Hypoxia signaling creates a more aggressive phenotype with increased potential for metastasis and decreased therapeutic efficacy. Carbonic anhydrase IX (CA9) functions as part of the cellular response to hypoxia by regulating intracellular pH to promote cell survival. Apurinic/Apyrimidinic Endonuclease-1-Reduction/oxidation Effector Factor 1 (APE1/Ref-1) is a multi-functional protein with two major activities: endonuclease activity in DNA base excision repair and a redox signaling activity that reduces oxidized transcription factors, enabling them to bind target sequences in DNA. APE1/Ref-1 is a central node in redox signaling, contributing to the activation of transcription factors involved in tumor survival, growth, and hypoxia signaling. This work evaluates the mechanisms underlying PDAC cell responses to hypoxia and APE1/Ref-1 redox signaling control of hypoxia inducible factor 1 alpha (HIF1a), a critical factor in hypoxia-induced CA9 transcription. We hypothesized that obstructing the HIF-CA9 axis at two points via APE1/Ref-1 inhibition and CA9 inhibition results in enhanced PDAC cell killing under hypoxic conditions. We found that HIF1a-mediated induction of CA9 is significantly attenuated following APE1/Ref-1 knock-down or redox signaling inhibition in patient-derived PDAC cells and pancreatic cancer-associated fibroblast cells. Additionally, dual-targeting of APE1/Ref-1 redox signaling activity and CA9 activity results in enhanced acidification and cytotoxicity of PDAC cells under hypoxic conditions as well as decreased tumor growth in an ex-vivo 3-dimensional tumor co-culture model. Further experiments characterized novel analogs of clinically relevant drugs targeting the key enzymes in this pathway, resulting in improved potency. These results underscore the notion that combination therapy is essential and demonstrate the potential clinical utility of blocking APE1/Ref-1 and CA9 function for novel PDAC therapeutic treatment.