KM-003

Amiloride improves mucociliary clearance (MC) by blocking airway epithelial sodium channels (ENaC) and expanding airway surface liquid (ASL). However, the low potency and rapid absorption of amiloride by airway epithelia translated into a short duration of efficacy as an aerosolized therapy for cystic fibrosis (CF) patients. To improve ENaC blocker CF pharmacotherapy, a more potent and durable ENaC blocker tailored for aerosol delivery was synthesized. Parion compound N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N'-4-[4-(2,3-dihydroxypropoxy)phenyl]butyl-guanidine methanesulfonate (552-02) was tested for potency and reversibility of ENaC block, epithelial absorption and biotransformation, selectivity, durability of ASL expansion under isotonic and hypertonic conditions in canine and human CF bronchial epithelial cells, and drug dissociation on ENaC in Xenopus oocytes. Short-circuit current assessed compound potency and reversibility, patch-clamp recordings of ENaC current assessed drug off-rate (k(off)), a gravimetric method and confocal microscopy measured mucosal water retention and ASL height, and drug absorption and biotransformation were assessed using liquid chromatography-mass spectrometry. Amiloride and 552-02 were tested in vivo for MC activity in sheep immediately and 4 to 6 h after aerosol dosing. Compared with amiloride, compound 552-02 was 60 to 100-fold more potent, it was 2 to 5-fold less reversible, it was slower at crossing the epithelium, and it exhibited a 170-fold slower k(off) value. 552-02 exhibited greater ASL expansion over 8 h in vitro, and it was more effective than amiloride at increasing MC immediately and 4 to 6 h after dosing. When combining hypertonic saline and 552-02, a synergistic effect on ASL expansion was measured in canine or CF bronchial epithelia. In summary, the preclinical data support the clinical use of 552-02 +/- hypertonic saline for CF lung disease.

In this study, a monoclonal antibody (mAb) termed SN6 was generated by immunizing a mouse with a non-T-cell leukemia antigen preparation isolated from cell membranes of leukemia cells derived from a patient (FJ) with non-T/non-B-cell-type acute lymphoblastic leukemia (ALL). SN6 was tested against a variety of cultured and uncultured human cell specimens by using a sensitive cellular radioimmunoassay. Among the 26 cultured malignant and nonmalignant cell lines tested, SN6 reacted with all of the 6 leukemic non-T/non-B (including pre-B)-cell lines tested--i.e., KM-3, NALM-16, REH, NALL-1, NALM-1, and NALM-6. Of these cell lines, 5 were derived from individual patients with ALL; the remaining 1 was from a patient with chronic myelocytic leukemia in blast crisis. In addition, SN6 reacted with 3 of 3 leukemic myelo-monocytic cell lines tested--i.e., ML-2, HL-60, and U937. SN6 did not react with any other cell lines. A consistent result was obtained with 42 fresh (uncultured) cell specimens derived from individual patients with several different types of leukemias. SN6 reacted with 11 of 16 non-T/non-B (including pre-B)-cell ALL specimens. In addition, it reacted with various myelo-monocytic leukemia cell specimens to various degrees. SN6 did not show a significant reaction with normal peripheral blood cells tested, which included B cells, T cells, granulocytes, monocytes, and erythrocytes. However, it reacted with a small population (approximately 1% as determined by immunofluorescence staining) of normal bone marrow cells. The approximate molecular mass of the glycoprotein antigen defined by SN6 was determined to be 160,000 by radioimmunoprecipitation followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Only one component of 80,000 daltons was formed upon reduction of the 160,000 molecular mass antigen. Therefore, this antigen is apparently a homodimer of a 80,000-dalton subunit. This conclusion was further corroborated by two-dimensional gel analysis, which showed a single well-defined spot for the reduced antigen. We designate this distinct human leukemia-associated cell surface antigen "GP160."