No description available.

<p>J1 (L-melphalanyl-L-<i>p</i>-fluorophenylalanyl ethyl ester) is a dipeptide derivative of the alkylating agent melphalan with increased cytotoxicity. In this thesis the preclinical pharmacology of J1 has been characterized.</p><p>Our results show that J1 rapidly enters the cells, where melphalan is released by hydrolysis. The maximum concentration (C_max) of melphalan was detected 15 min after exposure to J1 in human cancer cell lines. In comparison, melphalan exposure resulted in a 10-fold lower C_max that was shifted to later time points. J1 induced more DNA damage and apoptosis than melphalan. The cytotoxic activity and release of melphalan from J1 were inhibited by preincubating cells with the aminopeptidase inhibitor bestatin. In accordance with these results, we showed that J1 is a substrate for aminopeptidase N (APN), which may result in increased tumor selectivity.</p><p>J1 effectively inhibited cell growth in a set of neuroblastoma cell lines.<i> </i>Athymic mice carrying neuroblastoma xenografts were treated either with equimolar doses of melphalan or J1. J1 inhibited the tumor growth more effectively than melphalan and the untreated control, and was associated with higher caspase-3 activation, fewer proliferating tumor cells and decreased mean vascular density.</p><p>J1 and melphalan showed similar activity profiles when tested in 176 primary tumor cell cultures from patients, but J1 exhibited 50- to 100-fold higher potency. The difference was greater in some diagnoses (e.g. breast cancer, NHL and AML), and was exceptionally large in some breast cancer samples with aggressive phenotypes. A combination screening of J1 and standard chemotherapeutics yielded mostly additive interactions, except for etoposide which induced synergy in all tested cell lines.</p><p>In conclusion, the melphalan prodrug J1 is effectively transported into the cells, where aminopeptidases (for example APN) catalyze the formation of melphalan. J1 shows promising preclinical potential in the diagnoses neuroblastoma and breast cancer.</p>

J1 (L-melphalanyl-L-p-fluorophenylalanyl ethyl ester) is a dipeptide derivative of the alkylating agent melphalan with increased cytotoxicity. In this thesis the preclinical pharmacology of J1 has been characterized. Our results show that J1 rapidly enters the cells, where melphalan is released by hydrolysis. The maximum concentration (Cmax) of melphalan was detected 15 min after exposure to J1 in human cancer cell lines. In comparison, melphalan exposure resulted in a 10-fold lower Cmax that was shifted to later time points. J1 induced more DNA damage and apoptosis than melphalan. The cytotoxic activity and release of melphalan from J1 were inhibited by preincubating cells with the aminopeptidase inhibitor bestatin. In accordance with these results, we showed that J1 is a substrate for aminopeptidase N (APN), which may result in increased tumor selectivity. J1 effectively inhibited cell growth in a set of neuroblastoma cell lines. Athymic mice carrying neuroblastoma xenografts were treated either with equimolar doses of melphalan or J1. J1 inhibited the tumor growth more effectively than melphalan and the untreated control, and was associated with higher caspase-3 activation, fewer proliferating tumor cells and decreased mean vascular density. J1 and melphalan showed similar activity profiles when tested in 176 primary tumor cell cultures from patients, but J1 exhibited 50- to 100-fold higher potency. The difference was greater in some diagnoses (e.g. breast cancer, NHL and AML), and was exceptionally large in some breast cancer samples with aggressive phenotypes. A combination screening of J1 and standard chemotherapeutics yielded mostly additive interactions, except for etoposide which induced synergy in all tested cell lines. In conclusion, the melphalan prodrug J1 is effectively transported into the cells, where aminopeptidases (for example APN) catalyze the formation of melphalan. J1 shows promising preclinical potential in the diagnoses neuroblastoma and breast cancer.

Background: MY7 clinical trial compared dexamethasone plus melphalan (MD) vs. prednisone plus melphalan (MP) in multiple myeloma treatment and found no statistically significant difference in overall survival (OS) between the two groups. But, patients reacted to treatment differently. We aimed to identify patients who might have benefited from dexamethasone, and characterize them by their baseline demographic and clinical factors. Methods: First, the prognostic model for OS was developed on the MP arm. The estimated coefficients and baseline hazard were applied to the MD arm to derive martingale residuals (MR). Classification and regression tree analysis was done to identify independent predictive factors for OS and MR was used as response variable. All covariates in categorical shape were used as independent variables to develop the predictive model in MD arm. MP arm was divided accordingly. Subgroups with negative mean MR (survived > expected) were candidates for positive responders while those with positive mean MR (survived < expected) were candidates of negative responders. Mean MR in each subgroup and p values from comparison of OS (log rank test stratified by subgroups) were used to combine the appropriate subgroups as the positive responders or negative responders. Results: A total of 97 patients (42%) in MD arm were identified as positive responders and their OS (median of 44.5 months) was significantly longer than that (median of 33 months) in the corresponding subgroups in MP arm (HR = 0.56, 95% CI 0.4-0.8; p = 0.0014). All positive responders had three common baseline characteristics: aged ≤75 years, calcium concentration ≤2.6 mmol/L and Durie-Salmon stages 2 or 3. Among patients with ECOG performance status<2 those with either HGB≥100 mg/dl or HGB<100 mg/dl and WBC≥4,000 and <4 lytic bone lesions were categorized as positive responders. Also, among the patients with ECOG performance status≥2, males with >3 lytic bone lesions were positive responders. Negative responders (HR = 1.56, 95% confidence interval 1.1 – 2.2; p = 0.006) included patients aged >75 or aged ≤75 with calcium concentration >2.6 mmol/L or aged ≤75 with calcium concentration ≤2.6 mmol/L but had Durie-Salmon stage 1. Conclusions: Evaluation of the hypotheses validity warrants further studies. / Thesis (Master, Community Health & Epidemiology) -- Queen's University, 2008-07-21 13:46:53.748

Inhalation of chemical substances can cause irritation to airways and in high doses acute airway injury. When mice are exposed to the alkylating nitrogen mustard analogue melphalan they develop an acute airway inflammation with a rapid influx of neutrophils to the lungs. The acute phase is followed by long-term respiratory complications characterized by bronchitis, lung fibrosis, and airway hyperreactivity.      In this thesis, a mouse model for chemical airway inflammation was established and the effects on the lungs in a time span from 6 hours up to 3 months were investigated in order to study both acute effects and possible chronic injury. We find that treatment with corticosteroids, e.g. dexamethasone, effectively blocks the inflammatory reaction in several ways: Neutrophil influx to the lungs is diminished, the expression of the proinflammatory cytokines interleukin (IL) -6 and IL-1b is decreased and edema formation as well as development of lung fibrosis is mitigated. In acute airway inflammation we show that the antioxidant vitamin E can be used as a possible complement to corticosteroids but not as a replacement since it causes insufficient downregulation of the inflammatory response. We show the importance of the T lymphocytes as they play a prominent role in the pathogenesis of long-term lung injuries caused by melphalan. Especially the minor gd T cell subset is of major importance orchestrating a number of responses including the acute cytokine and neutrophil response and late-phase lung fibrosis. In order to find the critical time for dexamethasone treatment, mice were exposed to melphalan, treated with dexamethasone at specific time points and lung physiology and airway reactivity was measured in anaesthetized, tracheostomized mice using a small animal ventilator. From these results we conclude that an early treatment, i.e. within one hour after exposure, with dexamethasone is needed to prevent chronic lung injury.  This thesis was undertaken with the main goal to better understand the pathogenesis of melphalan-induced airway inflammation. We believe that our findings have shed new light in this area of research and hope that this increased knowledge may be of future clinical use.

The Fanconi Anemia (FA)/BRCA DNA damage repair pathway plays a critical role in the cellular response to stress induced by DNA alkylating agents and greatly influences drug response in cancer treatment. We recently reported that FA/BRCA DNA damage repair pathway genes are overexpressed and causative for resistance in multiple myeloma (MM) cell lines selected for resistance to melphalan. We hypothesized that the FA/BRCA DNA damage repair pathway mediates response and resistance to chemotherapeutic agents used to treat multiple myeloma and other cancers, and targeting this pathway is vital to overcoming drug resistance. In this dissertation, we show that FA/BRCA pathway genes are collectively overexpressed in MM, prostate, and ovarian cancer cell lines selected for resistance to melphalan and cisplatin, respectively. Interestingly, cells selected for resistance to topoisomerase II inhibitors selectively overexpress only FANCF. We also show that FA/BRCA pathway expression can be inhibited by the proteasome inhibitor bortezomib. FA/BRCA pathway mRNA expression was inhibited by bortezomib in myeloma cell lines and patient samples. FANCD2 gene and protein expression are downregulated by bortezomib, and remain attenuated in the face of melphalan treatment. Melphalan-induced FANCD2 foci formation was also inhibited by bortezomib, and this drug enhanced melphalan-induced DNA damage, likely via inhibition of FA-mediated DNA damage repair. Next, we analyzed regulation of the FA/BRCA pathway. We demonstrate that NF-kappaB, specifically the Re1B/p50 subunits, transcriptionally regulates members of the FA/BRCA pathway, and inhibition of these subunits by siRNA, BMS-345541, and bortezomib reduces FA/BRCA pathway expression. Furthermore, knocking down Re1B and p50 simultaneously attenuates FANCD2 protein expression and results in diminished DNA repair and enhanced sensitivity to melphalan. Importantly, melphalan resistance was restored when FANCD2 was re-expressed in these cells. We also show that bortezomib regulates FANCD2 protein expression directly, by inhibiting FANCD2 synthesis. Finally, we demonstrate that low-dose bortezomib arrests cells in G0/G1 and also overcomes the S-phase arrest induced by melphalan, likely via inhibition of ATR. Overall, our findings provide evidence for targeting the FA/BRCA pathway, either directly or indirectly, via inhibition of NF-kappaB or ATR, to enhance chemotherapeutic response and reverse drug resistance in multiple myeloma and other cancers.

<p>Oligopeptides can be used to carry cytotoxic agents in cancer chemotherapy, using tumour-associated proteins as the molecular target for selectivity. During the seventies and eighties Peptichemio, a cocktail of six alkylating oligopeptides carrying <i>m</i>-L-sarcolysin, was investigated in a wide variety of human malignancies. Positive clinical results were suggested to result from rapid and effective DNA-crosslinking following uptake in neoplastic cells, but also from antimetabolic properties of the drug. Although <i>m</i>-L-sarcolysin never reached widespread clinical use, the well established <i>para</i>-isomer melphalan still, after nearly fifty years, has a place in cancer chemotherapy.</p><p>The present study was undertaken to synthesise the melphalan containing analogue of the tripeptide P2 (L-prolyl-<i>m</i>-L-sarcolysyl-<i>p</i>-L-fluorophenylalanine ethyl ester, the main contributor to Peptichemio’s activity) and similar compounds, preferably dipeptides. The new compounds compared favourably with melphalan, <i>m</i>-L-sarcolysin and P2, considering their potency in vitro. Structure activity relationship analysis showed that the activity of melphalan dipeptides depended on the amino acid composition, sequence and end group modifications, but only to a minor degree on lipophilicity. Results suggested that the dipeptides, to exert their full cytotoxic activity, had to interact with specific biomolecules such as dipeptide transporters or peptidases. Although no active transport could be demonstrated the influence of peptide hydrolysis was obvious, thereby suggesting a rationale for increased activity as well as potential tumour selectivity in comparison with melphalan.</p><p>Preliminary in vivo studies in mice supported the results, despite equal alkylating capacity the dipeptide J1 (melphalanyl-<i>p</i>-L-fluorophenylalanine ethyl ester) and the tripeptide J3 (L-prolyl-melphalanyl-<i>p</i>-L-fluorophenylalanine ethyl ester) were more active than was melphalan on human tumour cells implanted in test animals although all drugs produced expected side effects, notably leukopenia, of similar magnitude.</p><p>In conclusion, oligopeptide derivatives of melphalan seem to provide improved cytotoxic activity and therapeutic index. Further development of such oligopeptides for clinical use seems worthwhile.</p>

Oligopeptides can be used to carry cytotoxic agents in cancer chemotherapy, using tumour-associated proteins as the molecular target for selectivity. During the seventies and eighties Peptichemio, a cocktail of six alkylating oligopeptides carrying m-L-sarcolysin, was investigated in a wide variety of human malignancies. Positive clinical results were suggested to result from rapid and effective DNA-crosslinking following uptake in neoplastic cells, but also from antimetabolic properties of the drug. Although m-L-sarcolysin never reached widespread clinical use, the well established para-isomer melphalan still, after nearly fifty years, has a place in cancer chemotherapy. The present study was undertaken to synthesise the melphalan containing analogue of the tripeptide P2 (L-prolyl-m-L-sarcolysyl-p-L-fluorophenylalanine ethyl ester, the main contributor to Peptichemio’s activity) and similar compounds, preferably dipeptides. The new compounds compared favourably with melphalan, m-L-sarcolysin and P2, considering their potency in vitro. Structure activity relationship analysis showed that the activity of melphalan dipeptides depended on the amino acid composition, sequence and end group modifications, but only to a minor degree on lipophilicity. Results suggested that the dipeptides, to exert their full cytotoxic activity, had to interact with specific biomolecules such as dipeptide transporters or peptidases. Although no active transport could be demonstrated the influence of peptide hydrolysis was obvious, thereby suggesting a rationale for increased activity as well as potential tumour selectivity in comparison with melphalan. Preliminary in vivo studies in mice supported the results, despite equal alkylating capacity the dipeptide J1 (melphalanyl-p-L-fluorophenylalanine ethyl ester) and the tripeptide J3 (L-prolyl-melphalanyl-p-L-fluorophenylalanine ethyl ester) were more active than was melphalan on human tumour cells implanted in test animals although all drugs produced expected side effects, notably leukopenia, of similar magnitude. In conclusion, oligopeptide derivatives of melphalan seem to provide improved cytotoxic activity and therapeutic index. Further development of such oligopeptides for clinical use seems worthwhile.

Η παρούσα μελέτη οδηγεί στην εξαγωγή συμπερασμάτων σχετικά με τη δραστικότητα στεροειδών εστέρων της Ν-ακετυλο-μελφαλάνης. Τα στοιχεία που προκύπτουν από την παραπάνω μελέτη δύνανται να συμπληρώσουν και να ενισχύσουν τα ήδη υπάρχοντα δεδομένα για τη συγκεκριμένη κατηγορία ενώσεων αφενός και, αφετέρου, να παρέχουν νέα δεδομένα για την αξιολόγηση και το μελλοντικό σχεδιασμό βελτιωμένων πιθανών στεροειδικών προφαρμάκων του συγκεκριμένου αλκυλιωτικού παραγοντα. Η βιολογική αξιολόγηση των νεοσύστατων ενώσεων περιλαμβάνει, στα πλαίσια της παρούσας εργασίας, την in vitro μελέτη τους όσον αφορά στην ικανότητα επαγωγής της Ανταλλαγής των Αδελφών Χρωματίδων (SCE) και αναστολής του Δείκτη Πολλαπλασιασμού (PRI), ως δείκτες της ενδεχόμενης in vivo αντιλευχαιμικής τους δράσης. Η μέτρηση της Ανταλλαγής των Αδελφών Χρωματίδων αποτελεί μία γρήγορη κι ευαίσθητη μέθοδο για την ανίχνευση παραγόντων που προκαλούν βλάβες στο γενετικό υλικό [210]. Η ικανότητα αποκοπής και επιδιόρθωσης διαφόρων τύπων αλλοιώσεων του DNA είναι μία γενικότερη ιδιότητα των ζωντανών κυττάρων, συμπεριλαμβανομένων και των καρκινικών. Η μη επιδιορθωμένη βλάβη, λοιπόν, εκφρασμένη σαν Ανταλλαγή των Αδελφών Χρωματίδων (SCE) σε φυσιολογικά κύτταρα, που έχει προκληθεί από συγκεκριμένες ενώσεις, υποδεικνύει ανάλογα την αδυναμία καρκινικών κυττάρων ζώντων οργανισμών να επιδιορθώσουν βλάβες που προκαλούνται από τους ίδιους παράγοντες. Η παραπάνω διατύπωση επαληθεύεται από πλήθος μελετών όπου η in vitro επαγωγή των SCEs συσχετίζεται άμεσα και σε περιπτώσεις, ανάλογα με την in vivo αντινεοπλασματική δραστικότητα ενώσεων της κατηγορίας των αλκυλιωτικών παραγόντων και των στεροειδικών εστέρων τους [165,173,174,197]. Αξιολογώντας τα αποτελέσματα της in vitro μελέτης, λοιπόν, διαπιστώνεται καταρχήν η περιορισμένη κυτταροτοξική ικανότητα της Ν-ακετυλο-μελφαλάνης έναντι του μη τροποποιημένου φαρμάκου, που εμφανίζεται περίπου 5 φορές περισσότερο δραστικό όσον αφορά την επαγωγή της SCE. Μελέτες, που αναφέρουν ότι η εισαγωγή της συγκεκριμένης τροποποίησης στο μόριο του φαρμάκου καθιστά τον αλκυλιωτικό παράγοντα πολύ λιγότερο τοξικό έναντι φυσιολογικών κυττάρων, καθιστούν την παραπάνω διαπίστωση αναμενόμενη. Σχολιάζοντας, περαιτέρω, τα αποτελέσματα που έδωσαν οι στεροειδείς εστέρες, είναι δυνατό να διατυπωθεί γενικότερα ότι η σύζευξη με τους στεροειδικούς σκελετούς αυξάνει σε όλες τις περιπτώσεις την ικανότητα πρόκλησης αλλοιώσεων στο γενετικό υλικό συγκριτικά με τον αλκυλιωτικό παράγοντα και, μάλιστα, σε ορισμένες από αυτές την καθιστά εφάμιλλη ή/και υπέρτερη σε σχέση με τη μη τροποποιημένη μελφαλάνη. Το γεγονός αυτό συνηγορεί με τα ως τώρα στοιχεία για τη θετική επίδραση του στεροειδικού φορέα. Εστιάζοντας, πιο συγκεκριμένα, στις διαφοροποιήσεις των στεροειδικών σκελετών, διαπιστώνεται καταρχήν ότι η εισαγωγή διπλού δεσμού μεταξύ 5 και 6 θέσης οδηγεί σε εμφανή ενίσχυση της δράσης των τελικών μορίων τόσο στην περίπτωση του σκελετού του ανδροστανίου όσο και στην περίπτωση του χολεστανίου. Περισσότερο αξιοσημείωτη διαφοροποίηση στη δράση, ωστόσο, εντοπίζεται στην περίπτωση του ανδροστανίου στη συγκέντρωση 0,4μΜ. Όσον αφορά στην εισαγωγή της NHCO ομάδας στο Δ δακτύλιο του απλού ανδροστανίου παρατηρείται αξιόλογη ενίσχυση της δράσης καταλήγοντας σε διπλασιασμό σχεδόν της γενοτοξικής δράσης του αρχικού εστέρα και παρέχοντας το δραστικότερο προϊόν. Αξιοσημείωτο παρουσιάζεται το γεγονός ότι σε σχέση με τον αλκυλιωτικό παράγοντα η επαγωγή των SCEs που προκλήθηκε από τον εστέρα με τον τροποιημένο στεροειδικό σκελετό είναι 6 φορές ισχυρότερη, ενώ ισχυρότερη εμφανίζεται και συγκριτικά με τη μελφαλάνη. Όλα τα παραπάνω στοιχεία ήταν αναμενόμενα στον αρχικό σχεδιασμό και επιβεβαιώνουν τα δεδομένα της βιβλιογραφίας. Όλες οι ενώσεις στην in vitro μελέτη χρησιμοποιήθηκαν σε δύο διαφορετικές συγκεντρώσεις. Με την αύξηση της δόσης παρατηρείται και ενίσχυση της δράσης των εστέρων με εξαίρεση το λακταμικό παράγωγο που η δράση του διατηρείται σχεδόν σταθερή. Κατά συνέπεια δύναται να επάγει τη δράση του σε μικρότερες δόσεις γεγονός που μπορεί να αποδειχτεί πλεονεκτικό σε in vivo χορήγηση. Όσον αφορά στην επίδραση των υπό μελέτη ενώσεων στο ρυθμό πολλαπλασιασμού των κυττάρων (PRI), διαπιστώνεται ότι ο PRI παραμένει στα ίδια περίπου επίπεδα με την ομάδα ελέγχου, κατά συνέπεια δεν παρατηρείται κυτταροστατική δράση. Τα παραπάνω συμπεράσματα έρχονται, σε γενικές γραμμές, σε συμφωνία με τα δεδομένα που έχουν παρουσιαστεί στο κεφάλαιο 4. Η σύζευξη του αλκυλιωτικού παράγοντα σε στεροειδικούς σκελετούς φαίνεται ότι εξυπηρετεί, πέρα από τη βελτίωση της μεταφοράς του κυτταροτοξικού παράγοντα, και την ενίσχυση της τελικής του δράσης, όπως διαπιστώνεται κυρίως με τον τροποποιημένο σκελετό. Πράγματι, η εισαγωγή της NHCO ομάδας ως ενδοκυκλικής λακτάμης οδήγησε σε παράγωγο με εμφανώς ενισχυμένη γενοτοξική ικανότητα. Η παραπάνω τροποίηση συνεισφέρει, αφενός, στην αύξηση της διαλυτότητας του τελικού μορίου στο ενδοκυτταρικό περιβάλλον, γεγονός που διευκολύνει την προσέγγιση του φαρμάκου στο θεραπευτικό στόχο, και, αφετέρου, αλληλεπιδρά ενδεχομένως με ανάλογες ομάδες που απαντώνται σε πρωτεΐνες και νουκλεϊκά οξέα. Επιπλέον, δεν παραλείπεται και η πιθανότητα ενζυμικές διεργασίες να μετατρέπουν τη συγκεκριμένη ομάδα του στεροειδικού σκελετού σε κυτταροτοξικά ενεργά ενδιάμεσα με συνέπεια τη συνεργική δράση στεροειδούς και αλκυλιωτικού παράγοντα. Η παραπάνω in vitro μελέτη σε φυσιολογικά λεμφοκύτταρα, βέβαια, παρέχει την ένδειξη για την ενδεχόμενη αντιλευχαιμική δράση. Μεγάλο ενδιαφέρον θα παρουσίαζε μία in vivo μελέτη σε καρκινικά μοντέλα. Είναι κατανοητό ότι εντός του οργανισμού το φάρμακο υπόκειται σε διάφορες μεταβολικές διεργασίες που απουσιάζουν από την in vitro μελέτη και, ταυτόχρονα, στο διαφοροποιημένο ενζυμικό προφίλ της νεοπλασίας, στοιχεία που πιθανά επηρεάζουν την τελική αντικαρκινική δράση. Συνυπολογίζοντας, λοιπόν, και τα αποτελέσματα μίας in vivo μελέτης μπορούμε να εξάγουμε πιο ασφαλή συμπεράσματα για τη σχέση χημικής δομής-βιολογικής δράσης και το μελλοντικό σχεδιασμό ανάλογων ενώσεων με βελτιωμένες ιδιότητες. / -

No description available.