Purpose The alkylating agent melphalan prolongs survival in multiple myeloma (MM) patients; however, it is associated with toxicities and development of drug-resistance. Here, we evaluated the efficacy of melphalan-flufenamide (Mel-flufen), a novel dipeptide prodrug of melphalan in MM. Experimental Design MM cell lines, primary patient cells, and the human MM xenograft animal model were utilized to study the antitumor activity of mel-flufen. Results Low doses of mel-flufen triggers a more rapid and higher intracellular concentrations of melphalan in MM cells than is achievable by free melphalan. Cytotoxicity analysis showed significantly lower IC50 of mel-flufen than melphalan in MM cells. Importantly, mel-flufen induces apoptosis even in melphalan-, and bortezomib-resistant MM cells. Mechanistic studies show that siRNA knockdown of aminopeptidase N, a key enzyme mediating intracellular conversion of mel-flufen to melphalan, attenuates anti-MM activity of mel-flufen. Furthermore, mel-flufen-induced apoptosis was associated with: 1) activation of caspases and PARP cleavage; 2) ROS generation; 3) mitochondrial dysfunction and release of cytochrome-c; and 4) induction of DNA damage. Moreover, mel-flufen inhibits MM cell migration and tumor-associated angiogenesis. Human MM xenograft studies showed a more potent inhibition of tumor growth in mice treated with mel-flufen than mice receiving equimolar doses of melphalan. Finally, combining mel-flufen with lenalidomide, bortezomib, or dexamethasone triggers synergistic anti-MM activity. Conclusion Our preclinical study supports clinical evaluation of mel-flufen to enhance therapeutic potential of melphalan, overcome drug-resistance, and improve MM patient outcome.

Recombinant molecules similar to the smallest active plasma-derived factor VIII molecule, a complex of an 80-kDa and a 90-kDa polypeptide chain lacking the B domain, have been produced using various factor VIII cDNA constructs in order to obtain primary translation products which were efficiently processed into the 80+90-kDa complex. Three types of single-chain cDNAs encoding B-domain-deleted derivatives factor VIII were designed, taking account of sites at Arg740 and Glu1649, assumed to be important for processing factor VIII.In the type 1 constructs, either Arg747, Arg752, or Arg776 in the N-terminal region of factor VIII B domain was fused to the N-terminus (Glu1649) of the 80-kDa subunit. In the type 2 construct r-VIII SQ, Ser743 was fused to Gln1638, creating a link of 14 amino acids between the C-terminus (Arg740) of the 90-kDa chain and N-terminus of the 80-kDa chain, whereas in type 2 r-VIII RH, Arg747 was fused to His1646. In the type 3 constructs, the B-domain was completely removed or replaced with 1-4 Arg residues.After expression in Chinese hamster ovary cells, the type 1 derivatives and the type 3 derivatives with 0-2 Arg residues inserted were found to be only partially processed and contained a large amount of the 170-kDa primary translation product. In contrast, most of the type 2 derivatives r-VIII SQ and r-VIII RH and the type 3 derivatives r-VIII R4 and r-VIII R5 containing three or four extra Arg residues preceding the N-terminus of the 80-kDa chain were processed into the desired 80+90-kDa chain complexes. The feature common to the most efficiently processed factor VIII deletion derivatives was that they contained the recognition motif for proteolytic cleavage by the membrane-bound subtilisin-like protease furin, which is expressed in most types of cells; that is, basic amino acid residues at positions -1 and -4 relative to the cleavage site at Glu1649. Biochemical studies of r-VIII SQ and r-VIII R5, two of the most effectively processed factor VIII derivatives, showed that both proteins had a normal factor VIII cofactor function, and had N-and C-termini of the 80-kDa and 90-kDa chains corresponding to those found in plasma-derived factor VIII.

SummaryThe pharmacokinetics of a second-generation recombinant B-domain deleted factor VIII (FVIII) preparation (r-VIII SQ) were studied in 36 patients with severe hemophilia A. In contrast to full-length recombinant FVIII, no albumin needs to be added to stabilize the final formulation of this B-domain deleted FVIII preparation.The in vivo recovery and half-life of r-VIII SQ were similar to those of plasma-derived (pd) FVIII (mean half-life of r-VIII SQ, 11.7 h). The volume of distribution and clearance were slightly, but significantly, higher for r-VIII SQ than for pdFVIII (p<0.05). Peak plasma levels of FVIII were consistently related to the administered dose of r-VIII SQ (r = 0.94, p<0.0001). The pharmacokinetic profile of r-VIII SQ remained essentially unchanged in a dose range of 25-100 IU/kg body weight and could be reproduced after repeated doses. r-VIII SQ was well tolerated.In conclusion, deletion of the B-domain of FVIII does not influence its in vivo pharmacokinetics.

The karyotypes of pristane-induced mouse plasmacytomas were studied by G banding. Only primary tumors or early passage generations were analyzed. In contrast to murine T cell leukemias that showed a regular trisomy of chromosome 15, all plasmacytomas showed a consistent translocation of the distal part of chromosome 15 to either chromosome 6 [rcpT(6;15)] or 12 [T(12;15)]. The specific breakpoints were at 6C, 15D3/E ro D2/3 and 12F2. Early passage generations often showed a mixed population with two different translocations, suggesting polyclonal origin. Considered together with the known karyotypic features of murine and human lymphomas, these findings support the theory that the nonrandom chromosomal changes in lymphoproliferative malignancies are associated with the type of the target cell, rather than with the etiological agent. Moreover, the involvement of the chromosomes known to carry the heavy chain (12) and the light chain (6) determinants, respectively, raises the question of whether the translocations may be related to the DNA level rearrangements known to occur during the differentiation of normal plasma cells.

IntroductionProphylactic replacement therapy for hemophilia A is based on intravenous infusions of purified factor VIII (FVIII) concentrates. [1][2][3][4][5][6][7][8][9][10] Since the half-life of human FVIII is about 10 to 12 hours, 11 infusions typically need to be repeated every 2 to 3 days to maintain a FVIII level above 1% in patients treated according to the pharmacokinetic (PK) dosing model. 1,[12][13][14][15] The prevention of bleeding episodes, especially in young patients, is of vital importance, as it reduces the occurrence of hemophilic arthropathy, which usually develops secondary to repeated intraarticular bleeding episodes. 3,4,6,7,[16][17][18][19][20][21] Prophylactic infusions performed in order to prevent bleeding episodes can delay the time of onset of hemophilic arthropathy and reduce the severity of pain and sequelae. Patients dosed along PK parameters usually demonstrate very few bleeding episodes and low morbidity. 22 Repeated regular intravenous infusions performed at home every 2 to 3 days over the course of decades places a great burden on patients to be compliant with their therapy. Compliance with treatment directives has been directly linked to the number of infusions given, with compliance increasing as the number of infusions decreased. 23 A FVIII molecule or product formulation that demonstrates increased time in circulation could greatly improve the efficacy and quality of life associated with prophylactic treatment for hemophilia A.The method most commonly used for prolongation of half-life of recombinant proteins, the covalent incorporation of polyethylene glycol (PEG; PEGylation), 24 is not yet available for FVIII. An alternative approach is to incorporate the FVIII protein with a carrier molecule that can be modified by PEGylation, resulting in a prolongation of the time the molecule provides hemostatic efficacy. The advantage with such an approach is that the FVIII molecule would not need to be altered and, provided that the carrier did not confer any conformational changes to the FVIII or act as an adjuvant for the immune system, the risk for FVIII inhibitor development would not be increased compared with standard treatment. Furthermore, since low levels of FVIII have been shown to confer prophylactic efficacy, the goal of maintaining FVIII levels above 1% as in the PK model may not be necessary. [25][26][27][28] Baru et al 29 reported the use of PEGylated liposomes as carriers for recombinant FVIII (Kogenate FS). Synthetic PEGylated liposomes, composed of 90% (wt/wt) palmitoyl-oleoylphosphatidylcholine (POPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N- [poly-(ethyleneglycol) Patients, materials, and methods Study designThe study was conducted in accordance with International Conference on Harmonization Good Clinical Practice (ICH GCP) guidelines. Approval was obtained from the appropriate ethics committees as well as by the Ministry of Health. All patients gave written informed consent prior to participation. The study was designed as a controlled, patient-bl...

BackgroundMotor function assessments with rating scales in relation to the pharmacokinetics of levodopa may increase the understanding of how to individualize and fine-tune treatments.ObjectivesThis study aimed to investigate the pharmacokinetic profiles of levodopa-carbidopa and the motor function following a single-dose microtablet administration in Parkinson’s disease.MethodsThis was a single-center, open-label, single-dose study in 19 patients experiencing motor fluctuations. Patients received 150% of their individual levodopa equivalent morning dose in levodopa-carbidopa microtablets. Blood samples were collected at pre-specified time points. Patients were video recorded and motor function was assessed with six UPDRS part III motor items, dyskinesia score, and the treatment response scale (TRS), rated by three blinded movement disorder specialists.ResultsAUC0–4/dose and C max/dose for levodopa was found to be higher in Parkinson’s disease patients compared with healthy subjects from a previous study, (p = 0.0008 and p = 0.026, respectively). The mean time to maximum improvement in sum of six UPDRS items score was 78 min (±59) (n = 16), and the mean time to TRS score maximum effect was 54 min (±51) (n = 15). Mean time to onset of dyskinesia was 41 min (±38) (n = 13).ConclusionsIn the PD population, following levodopa/carbidopa microtablet administration in fasting state, the Cmax and AUC0–4/dose were found to be higher compared with results from a previous study in young, healthy subjects. A large between subject variability in response and duration of effect was observed, highlighting the importance of a continuous and individual assessment of motor function in order to optimize treatment effect.

The pertubation treatment significantly enhanced the clinical pregnancy rate and was well tolerated. No complications were noted. The combined treatment of clomiphene citrate, pertubation and insemination can be used as a cost-effective, first-line treatment for couples with unexplained infertility.

In conclusion, melflufen can safely be given to cancer patients, and the toxicity profile was as expected for alkylating agents; RPTD is 50 mg Q3W. Reversible and manageable bone marrow suppression was identified as a DLT. Clinical activity is suggested in ovarian cancer, but modest activity in treatment of refractory NSCLC.