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9-(2-Phosphonylmethoxyethyl)-N6-cyclopropyl-2,6-diaminopurine (cpr-PMEDAP) is an acyclic nucleotide analog of the [9-(2-phosphonylmethoxyethyl)-] (PME) series containing a cyclopropyl substituent on the N6 position of the 2,6-diaminopurine (DAP) base. Growth inhibition assays in a broad range of tumor cell lines demonstrated that this analog had potent antiproliferative activity with IC50 values similar to those of the structurally related guanine analog 9-(2-phosphonylmethoxyethyl)guanine (PMEG). A substantially lower growth inhibitory effect was observed for the 2,6-diaminopurine analog, PMEDAP. To dissect the basis for these varying potencies, the metabolism of the three analogs was examined in a human pancreatic carcinoma cell line, BxPC-3. HPLC analysis of the intracellular metabolites demonstrated that the cpr-PMEDAP was deaminated to PMEG and subsequently phosphorylated to PMEG mono- and diphosphates (PMEGp and PMEGpp). The level of PMEGpp generated from cpr-PMEDAP-treated cells was 50% greater than the level generated from cells incubated with PMEG. The presence of PMEG in the DNA of cells incubated with cpr-PMEDAP confirmed that the cpr-PMEDAP was converted to PMEG. In contrast, PMEDAP was not deaminated to PMEG, but directly phosphorylated to PMEDAPp and PMEDAPpp. The adenylate deaminase inhibitor 2'-deoxycoformycin (dCF) inhibited the conversion of cpr-PMEDAP in a rat liver cytosolic extract and increased the IC50 value for growth inhibition by 40-fold. The antiproliferative activities of PMEG and PMEDAP were unaffected by dCF. Thus, it appears that cpr-PMEDAP, but not PMEDAP, is converted by an adenylate deaminase-like enzyme and functions as a prodrug of PMEG.

A new class of thrombin inhibitors based on sequence-specific single-stranded DNA oligonucleotides (thrombin aptamer) has recently been identified. The aptamer-binding site on thrombin was examined by a solid-phase plate binding assay and by chemical modification. Binding assay results demonstrated that the thrombin aptamer bound specifically to alpha-thrombin but not to gamma-thrombin and that hirudin competed with aptamer binding, suggesting that thrombin's anion-binding exosite was important for aptamer-thrombin interactions. To identify lysine residues of thrombin that participated in the binding of the thrombin aptamer, thrombin was modified with fluorescein 5'-isothiocyanate in the presence or absence of the thrombin aptamer, reduced, carboxymethylated, and digested with endoproteinase Arg-C. The digestion products were analyzed by reversed-phase high performance liquid chromatography and the peptide maps compared. Four peptides with significantly decreased modification in the presence of the aptamer were identified and subjected to N-terminal sequence analysis. Results indicated that B chain Lys-21 and Lys-65, both located within the anion-binding exosite, are situated within or in close proximity to the aptamer-binding site of human alpha-thrombin. The thrombin aptamer binds to the anion-binding exosite and inhibits thrombin's function by competing with exosite binding substrates fibrinogen and the platelet thrombin receptor.

We have purified a cDNA fragment complementary to the mRNA coding for one of the major urinary proteins (MUPs) synthesized in the mouse liver. Using this cDNA as a hybridization probe, we have shown that the level of MUP mRNA is lower in the livers of females and castrated males than in those of males. The addition of testosterone to females and castrated males results in an increase in the concentration of the mRNA to levels found in males. There are approximately 15 gene per haploid genome coding for the MUPs; this allows a possible new interpretation of some of the genetic data concerning the regulation of levels of the different MUPs in the urine (Szoka and Paigen, 1978). Finally, we have shown that mouse MUP and rat alpha 2u-globulin mRNA share common sequences, but that there are surprising differences in gene number and regulation of the genes in these two closely related animals.

SUMMARY It is unfortunate that we cannot report, in the area of coagulation, advances that have been seen in related fields such as thrombolytic therapy. The reported progress (Gold et al, 1984; Van de Werf et al, 1984) with human recombinant tissue plasminogen activator (Pennica et al, 1983) augers well for the application of recombinant technology to the problems faced by patients with coagulation defects. While plasminogen activator is being assessed in an acute therapeutic setting, its use signals a beginning of the application of the technology to abnormalities of the haemostatic mechanism. Chronic administration of coagulation factors for prophylaxis and replacement therapy would appear to be just one more step down the pathway illuminated by the biochemists, microbiologists and cell biologists who have preceded the clinicians in this promising area. There is no record of the use of genetically engineered materials in the treatment of coagulation defects, primarily because the body of knowledge and refined techniques have only recently been acquired. For this reason we have had to project developments in other areas onto the problems that exist for the haemostatically compromised patient. In describing the potential usefulness of these technologies, it is difficult to ascertain where the logical projection, from a fully investigated model system, diverges from flights of imaginative fancy. Cloning projects considered overly ambitious and grandiose at the beginning of this decade are already accomplished feats. The feasibility of gene therapy in the mammalian system has been demonstrated, and trade publications now discuss governmental approval for investigative use of this procedure in 1985. Panels of physicians, scientists and even politicians now seriously contemplate and promulgate views and regulations pertaining to the efficacy and ethics of the use of genetic engineering in the treatment of human disease. The haemophilias will certainly be among the first genetic diseases to be approached with the techniques of recombinant DNA technology. Diagnostic testing, using cloned DNA, is already underway and therapeutic trials are predicted for the near future. Every observer of this rapidly growing field has to define for himself when the future is. For the potential carrier of haemophilia B, the future is now. For the physician managing the patient with a haemophilic inhibitor, the future can't come soon enough. And, for those who are concerned with man's tampering with the gene pools of living things, from viruses to humans, the future comes too quickly to be dealt with in a rational and understanding way.