Philip E. Morris

Potent orally bioavailable purine nucleoside phosphorylase inhibitor BCX-4208 induces apoptosis in B- and T-lymphocytes—A novel treatment approach for autoimmune diseases, organ transplantation and hematologic malignancies

The profound suppression of T-cell immunity seen in purine nucleoside phosphorylase (PNP; EC 2.4.2.1) deficient patients supports potential application of inhibitors of PNP in the therapy of T-cell mediated diseases. BCX-4208 is a novel potent transition state analog inhibitor of human PNP with an IC(50) of 0.5 nM. PNP inhibition leads to elevation of dGuo which is converted to dGTP mainly in lymphocytes causing imbalance in deoxynucleotide (dNTP) pools and cell apoptosis. In in vitro studies, neither BCX-4208 nor dGuo alone inhibits proliferation of lymphocytes. BCX-4208 in the presence of 10 microM deoxyguanosine (dGuo) inhibits lymphocyte proliferation induced by MLR, IL-2 or Con A with IC(50)s of 0.159, 0.26 and 0.73 microM, respectively. The IC(50) for dGuo in the presence of 1 microM BCX-4208 for the IL-2 stimulated lymphocytes was 3.12 microM. dGTP in human lymphocytes is elevated and a 3-5 fold increase in dGTP results in 50% inhibition after in vitro exposure to BCX-4208 and dGuo. Flow cytometric analyses of human lymphocytes using annexin V staining reveal that BCX-4208 in the presence of dGuo induces cellular apoptosis in T-cells (CD3+), B-cells (CD20+, CD19+) and NK (CD56+) cells. BCX-4208 is orally bioavailable in mice and elevates plasma dGuo levels to 3.7 microM (predose levels<0.004 microM), similar to levels seen in PNP-deficient patients and levels needed to cause apoptosis in T and B-cells. These data support the evaluation of BCX-4208 in the treatment of T-cell and B-cell mediated diseases. BCX-4208 is currently undergoing early clinical investigation in psoriasis and gout.

In vivo metabolism of α,α,β,β-tetradeutero-n, N-dimethyltryptamine in rodent brain

The metabolism of alpha,alpha,beta,beta- tetradeutero -N,N -dimethyltryptamine ( D4DMT ) in rat brain in vivo as a function of time and dose was examined. Quantification of D4DMT and its respective deutero-metabolites was accomplished using gas chromatographic/mass spectrometric/selected ion monitoring/isotope dilution techniques. The results of this study indicate that D4DMT is metabolized to the corresponding deutero-N-methyltryptamine, tryptamine, 1,2,3,4-tetrahydro-beta-carboline, and 2-methyl-1, 2,3,4-tetrahydro-beta-carboline in rat brain. The subcellular distribution of D4DMT and the aforementioned metabolites is also reported.

Comparison of the brain levels of N,N-dimethyltryptamine and α,α,β,β-tetradeutero-N,N-dimethyltryptamine following intraperitoneal injection: The IN VIVO kinetic isotope effect

A comparison of the brain levels (microgram/g wet weight of tissue) of the hallucinogen N,N-dimethyltryptamine (DMT) and its deuterated analog alpha, alpha, beta, beta-tetradeutero-DMT (D4DMT) as a function of time and dose is reported. It was observed that the presence of deuterium in the alpha- and beta-positions of the ethylamine side-chain led to a potentiation of the level of DMT in brain. Strikingly different dynamics of uptake and clearance were also noted. We propose that these results are due to primary kinetic isotope effect, illustrating the importance of the alpha-position in the metabolism of DMT.

Ontogeny of N,N-Dimethyltryptamine and related indolealkylamine levels in neonatal rats

The present study deals with the measurement of the brain levels of the two potent hallucinogens N,N-dimethyltryptamine (DMT) and 5-methoxy-N,N-dimethyltryptamine (OMB), the biogenic amine tryptamine (TA), and its condensation product 1,2,3,4-tetrahydro-beta-carboline (THBC) in rats of various ages. Using gas chromatography-mass spectrometry with isotope dilution, we detected DMT, OMB, and THBC in neonatal rats from birth. DMT levels remained low until days 12 and 17 at which time they increased significantly and then returned to the initial low levels for all subsequent ages. The levels of OMB were higher than those measured for DMT with the highest levels being observed at days 12 and 17, and also on day 31. However, the levels for OMB showed much more variation. Although elevated levels of DMT and OMB have been correlated with stress, there are no known functions for these compounds. TA levels remained below detection limits until day 19. THBC levels were observed to be highest on days 22 and 31. The role that THBC plays in mammalian tissues is not known.

Synthesis of labeled BCX-4208, a potent inhibitor of purine nucleoside phosphorylase.

BCX-4208, a novel inhibitor of the enzyme purine nucleoside phosphorylase, mimics the charged ribosyl oxocarbenium ion formed during the transition state of the enzyme-catalyzed C-N bond cleavage of nucleosides. A slow-onset, tight-binding inhibitor with a Ki(*) of 16 +/- 1.4 pM, BCX-4208 is one of the most potent inhibitors known for the enzyme. In support of our BCX-4208 clinical program, a mass spectrometric assay has been developed that required labeled BCX-4208 as an internal standard. The synthesis of [(2)H](2)-BCX-4208 and [(13)C]-BCX-4208 is described in this report.

Synthesis of deuterated-BCX-34 (peldesine)

BCX-34 (peldesine) is a novel purine nucleoside phosphorylase inhibitor that is in human clinical trials for the treatment of T-cell cancers and for HIV infected patients. In support of our BCX-34 clinical program, a mass spectrometric assay has been developed utilizing a deuterated-BCX-34 analog as an internal standard. The synthesis of tetra-deuterated-BCX-34 (peldesine) from ethyl nicotinate (2,4,5,6-[2H]4) is described in this report. Copyright

Facilitation of an operant task in the rat following injection of whole brain extract

It has been shown that the administration of trained donor brain extract to naive rats results in facilitation of performance on the same task. In the present study a group of food deprived rats was trained to press a lever for food on a continuous schedule of reinforcement until they reached criterion. The animals were then sacrificed, their brains excised, homogenized and the small proteins (m.w. less than 3500) extracted. A group of untrained rats was also sacrificed and their brains extracted. Three groups of rats were used as recipients, receiving either trained donor or untrained donor brain extract or saline. The animals were tested individually for one-hour sessions at 18, 42 and 66 hours after the injection. The number of bar presses made by each rat was noted and the group mean plus or minus the standard deviation were calculated for each session. The results of a one-way analysis of variance showed that the group which received trained donor brain extract performed at a higher rate than either control group. These data suggest that some factor, (specific or non-specific), associated with the task has been transferred.