Discovery of tert-Butyl Ester Based 6-Diazo-5-oxo-l-norleucine Prodrugs for Enhanced Metabolic Stability and Tumor Delivery.

The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) exhibits remarkable anticancer efficacy; however, its therapeutic potential is hindered by its toxicity to gastrointestinal (GI) tissues. We recently reported the discovery of DRP-104, a tumor-targeted DON prodrug with excellent efficacy and tolerability, which is currently in clinical trials. However, DRP-104 exhibits limited aqueous solubility, and the instability of its isopropyl ester promoiety leads to the formation of an inactive M1-metabolite, reducing overall systemic prodrug exposure. Herein, we aimed to synthesize DON prodrugs with various ester and amide promoieties with improved solubility, GI stability, and DON tumor delivery. Twenty-one prodrugs were synthesized and characterized in stability and pharmacokinetics studies. Of these, P11, tert-butyl-(S)-6-diazo-2-((S)-2-(2-(dimethylamino)acetamido)-3-phenylpropanamido)-5-oxo-hexanoate, showed excellent metabolic stability in plasma and intestinal homogenate, high aqueous solubility, and high tumor DON exposures and preserved the ideal tumor-targeting profile of DRP-104. In conclusion, we report a new generation of glutamine antagonist prodrugs with improved physicochemical and pharmacokinetic attributes.

Tumor-Targeted Delivery of 6-Diazo-5-oxo-l-norleucine (DON) Using Substituted Acetylated Lysine Prodrugs.

6-Diazo-5-oxo-l-norleucine (DON) is a glutamine antagonist with robust anticancer efficacy; however, its therapeutic potential was hampered by its biodistribution and toxicity to normal tissues, specifically gastrointestinal (GI) tissues. To circumvent DON's toxicity, we synthesized a series of tumor-targeted DON prodrugs designed to circulate inert in plasma and preferentially activate over DON in tumor. Our best prodrug 6 (isopropyl 2-(6-acetamido-2-(adamantane-1-carboxamido)hexanamido)-6-diazo-5-oxohexanoate) showed stability in plasma, liver, and intestinal homogenates yet was readily cleaved to DON in P493B lymphoma cells, exhibiting a 55-fold enhanced tumor cell-to-plasma ratio versus that of DON and resulting in a dose-dependent inhibition of cell proliferation. Using carboxylesterase 1 knockout mice that were shown to mimic human prodrug metabolism, systemic administration of 6 delivered 11-fold higher DON exposure to tumor (target tissue; AUC0- t = 5.1 nmol h/g) versus GI tissues (toxicity tissue; AUC0- t = 0.45 nmol h/g). In summary, these studies describe the discovery of a glutamine antagonist prodrug that provides selective tumor exposure.

Reviving Lonidamine and 6-Diazo-5-oxo-L-norleucine to Be Used in Combination for Metabolic Cancer Therapy.

Abnormal metabolism is another cancer hallmark. The two most characterized altered metabolic pathways are high rates of glycolysis and glutaminolysis, which are natural targets for cancer therapy. Currently, a number of newer compounds to block glycolysis and glutaminolysis are being developed; nevertheless, lonidamine and 6-diazo-5-oxo-L-norleucine (DON) are two old drugs well characterized as inhibitors of glycolysis and glutaminolysis, respectively, whose clinical development was abandoned years ago when the importance of cancer metabolism was not fully appreciated and clinical trial methodology was less developed. In this review, a PubMed search using the words lonidamine and 6-diazo-5-oxo-L-norleucine (DON) was undertaken to analyse existing information on the preclinical and clinical studies of these drugs for cancer treatment. Data show that they exhibit antitumor effects; besides there is also the suggestion that they are synergistic. We conclude that lonidamine and DON are safe and potentially effective drugs that need to be reevaluated in combination as metabolic therapy of cancer.

Affinity of antineoplastic amino acid drugs for the large neutral amino acid transporter of the blood-brain barrier.

The relative affinity of six anticancer amino acid drugs for the neutral amino acid carrier of the blood-brain barrier was examined in rats using an in situ brain perfusion technique. Affinity was evaluated from the concentration-dependent inhibition of L-[14C]-leucine uptake into rat brain during perfusion at tracer leucine concentrations and in the absence of competing amino acids. Of the six drugs tested, five, including melphalan, azaserine, acivicin, 6-diazo-5-oxo-L-norleucine, and buthionine sulfoximine, exhibited only low affinity for the carrier, displaying transport inhibition constants (Ki, concentrations producing 50% inhibition) ranging from 0.09 to 4.7 mM. However, one agent - D,L-2-amino-7-bis[(2-chloroethyl)amino]- 1,2,3,4-tetrahydro-2-naphthoic acid (D,L-NAM) - demonstrated remarkably high affinity for the carrier, showing a Ki value of approximately 0.2 microM. The relative affinity (1/Ki) of D,L-NAM was greater than 100-fold that of the other drugs and greater than 10-fold that of any compound previously tested. As the blood-brain barrier penetrability of most endogenous neutral amino acids is related to their carrier affinity, the results suggest that D,L-NAM may be a promising agent which may show enhanced uptake and distribution to brain tumors.

Pharmacokinetic and phase I study of intravenous DON (6-diazo-5-oxo-L-norleucine) in children.

DON (6-diazo-5-oxo-L-norleucine), a glutamine antagonist, has been subjected to limited clinical trials since 1957. Use of the drug in adults has been curtailed due to sparse reports of effectiveness as well as its dose-limiting toxicities, i.e., severe nausea, vomiting and mucositis. In earlier studies, children given DON orally in combination with 6-mercaptopurine had significant prolongation of remission of acute leukemias during maintenance therapy. As DON is acid-labile and relatively unstable in solution, oral administration does not appear to be ideal for DON. In the trial described in this report, i.v. DON therapy was studied, using i.v. chlorpromazine to control vomiting, in 20 children, 17 of whom were evaluable following treatment at DON dose levels ranging from 150 mg/m2 to 520 mg/m2. Nausea and vomiting, the dose-limiting toxicity for adults, was controlled with chlorpromazine. Mucositis, which has also been observed in adults, did not occur in the children given DON i.v. A maximum tolerated dose was not defined; however, the projected maximum tolerated dose appears to be in excess of 450 mg/m2. DON was measured in plasma using a rapid-sampling HPLC procedure. The total body clearance, plasma t1/2, and area under the plasma concentration curve (AUC) were calculated using a noncompartmental method. The drug is rapidly cleared from plasma (t 1/2 = 3 h), and its volume of distribution is approximately twice that of total body water in children. These pharmacokinetic data, differ from that of adults reported by others. Specifically, the plasma t 1/2 for children is longer: total body clearance (Cl), and volume of distribution at steady state (Vss) are greater. In addition, no dose dependency of t 1/2, Cl or Vss was observed in this study, and the DON pharmacokinetics were linear and predictable. Five of nine children with acute leukemia showed improvement, though insufficient for classification as partial response, and five of eight children with solid tumors also showed improvement. Further trials using DON in combination with thiopurines or other agents appear indicated.