Multifaceted impact of adipose conditioned media: Obesity-driven promotion of prostate cancer and cancer stem cell dynamics.

Obesity is an established risk factor for the development and progression of prostate cancer (PC). This study used adipose conditioned media (ACM) from differentiated adipocytes to assess its effect on PC development and aggressiveness. Due to limited research on ACM's impact on isolated PC stem cells (PCSCs), we also examined CD44+ PCSCs. ACM notably boosted interleukin-1ß (IL-1ß), IL-6, and IL-8 production in normal prostate epithelial cells and LNCaP cells. It also increased IL-6 and IL-8 production in PC3 and CD44+ LNCaP cells, and IL-1ß and IL-6 production in CD44+ PC3 cells. This indicates that ACM induces the production of inflammatory cytokines in both cancer and prostate epithelial cells. Furthermore, ACM promoted proliferation in androgen receptor (AR)-negative PC3 cells, CD44+ PC3 PCSCs, and nonmalignant RWPE cells, without affecting AR-positive LNCaP cells. In addition, ACM-enhanced invasion and migration potential in both PC3 and CD44+ PC3 cells. Western blot analysis indicated the involvement of NF-κB and AKT pathways in ACM-induced proliferation in PC3 cells and NF-κB in PCSCs. In ACM-treated PC3 cells, E-cadherin was downregulated, while N-cadherin, Snail, vimentin, fibronectin, and Twist were upregulated, suggesting ACM-induced invasion via classical epithelial-to-mesenchymal transition (EMT) pathways. In response to ACM, PCSCs exhibited increased expression of E-cadherin, Snail, and vimentin, which are partial EMT markers promoting stemness and resistance to apoptosis. In addition, increased expressions of Nanog, Oct3/4, survivin, and Bcl-2 were observed. Although the molecules we studied have diverse effects on cellular regulation, our data emphasize obesity's multifaceted role in promoting and aggressing PC, notably affecting PCSC populations.

Association Between Plasma Asprosin Levels and Gestational Diabetes Mellitus.

Purpose: This study sought to investigate whether asprosin can be used in the diagnosis of GDM or for diagnostic purposes in high-risk pregnancies, along with a review of other parameters that may be associated with serum asprosin levels. Patients and Methods: The study investigated the association between gestational diabetes mellitus (GDM) and asprosin levels. A total of 93 participants; 30 patients with GDM, 33 healthy pregnant women with normal glucose tolerance (NGT), and 30 healthy non-diabetic women (control group) at the Endocrinology and Metabolic Diseases outpatient clinic of a tertiary care university hospital were enrolled in the study. Patients with GDM and NGT were examined in terms of GDM between the 24th and 28th week of pregnancy (2nd trimester). Patient data were collected during routine examinations, and asprosin levels were measured using the ELISA method. All participants underwent testing for measurements of serum hemoglobin, insulin, C-peptide, fasting plasma glucose, and glycated hemoglobin (HbA1c) levels following a fasting period of at least eight hours. Results: Asprosin levels were higher in pregnant women with NGT and with GDM versus controls (Control-NGT asprosin, p = 0.001; Control-GDM asprosin, p = 0.001). Pregnant women with GDM had higher asprosin levels than those with NGT (p = 0.001). In detecting GDM in pregnant women, an asprosin cutoff value of >31.709 ng/mL yielded a sensitivity of 93.3%, specificity of 90.9%, positive predictive value of 90.3%, and negative predictive value of 93.75% (p < 0.001). Conclusion: Serum asprosin levels can potentially be used as a marker in the diagnosis of GDM.

A novel biomarker in acute cholecystitis: YKL-40.

BACKGROUND: The lack of a specific biomarker that can be used in the diagnosis of acute cholecystitis, a common cause of admission to the emergency department, delays physician efforts to diagnose and treat these patients. Therefore, the aim of this study was to measure plasma YKL-40 levels and investigate their diagnostic value in patients with acute cholecystitis (AC). METHODS: This study was carried out between February 2020 and September 2020 in the adult emergency department of a tertiary university hospital. Permission was obtained from the Ethics Committee of Scientific Research on 03/02/2020 with Decision No. 03/16. The study included 80 patients who were diagnosed with acute cholecystitis and 80 healthy volunteers without known chronic diseases. RESULTS: The median YKL-40 protein level was 798.66 pq/mL in the patient group and 392.45 pq/mL in the control group. A statistically significant difference in YKL-40 protein levels was found between the two groups. YKL-40 protein levels were significantly higher in patients diagnosed with acute cholecystitis than in healthy individuals (p < 0.001). A positive correlation was found between YKL-40 protein levels and ALT, AST, LDH, and GGT levels (r = 0.272, p = 0.015; r = 0.397, p < 0.001; r = 0.386, p < 0.001; and r = 0.264, p = 0.018; respectively). CONCLUSION: When evaluated together with physical examination, radiological imaging and other laboratory parameters, we think that plasma YKL-40 levels can be used effectively in the diagnosis of acute cholecystitis.

The synergistic anticancer effect of salinomycin combined with cabazitaxel in CD44+ prostate cancer cells by downregulating wnt, NF-κB and AKT signaling.

BACKGROUND: Tumor-initiating or cancer stem cells (CSCs) reduce the effectiveness of conventional therapy. Thus, it is crucial to eliminate CSCs while killing bulky cancer cells using a combination of conventional chemotherapy and anti-CSC drugs. Salinomycin is a selective inhibitor against CSCs and shows promise in combination applications. The aim of the study was to examine the efficacy of co-administered cabazitaxel and salinomycin on the survival of prostate cancer cells and CSCs. METHODS AND RESULTS: CD44 + stem cells were isolated from human PC3 prostate cancer cells by using magnetic activated cell sorting. The cells were concomitantly exposed to salinomycin and cabazitaxel, and the cell survival was determined by MTT test. Apoptosis was assessed by image-based cytometer, and cell migration was evaluated by wound healing assay. The expression of target mRNA and protein were assessed by RT-qPCR and Western blot, respectively. Combination index (CI) analysis showed that simultaneous administration of salinomycin and cabazitaxel was able to exert strong synergistic effect on CD44 + subpopulation (CI = 0.33), but no synergism was observed in PC3 cells. The combination of the two agents significantly increased Bax, cytochrome c, caspase-3 and - 8 mRNA expression in CD44 + CSCs, causing apoptosis. The applied therapy strategy strongly inhibited the phosphorylation of Akt, protein expression of Akt1, NF-κB and Wnt. CONCLUSIONS: In conclusion, our data suggest that combining salinomycin with cabazitaxel shows promise as a prostate cancer treatment approach that can target CSCs.

Midkine silencing enhances the anti-prostate cancer stem cell activity of the flavone apigenin: cooperation on signaling pathways regulated by ERK, p38, PTEN, PARP, and NF-κB.

Prostate cancer (PCa) is the most common cancer in men worldwide. Midkine (MK) is overexpressed in PCa, as well as in tumor-initiating cells termed cancer stem cells (CSCs). Apigenin is a dietary flavone with considerable anti-tumor activities. In this study, we explored the possible therapeutic use of MK silencing, apigenin treatment, and a combination of both on human PCa and prostate cancer stem cells (PCSCs). CD44+CD133+ PC3 and CD44+ LNCaP CSCs were isolated from their parent cell lines. Both MK knockdown and apigenin treatment resulted in loss of cell viability in PCSCs, and these effects were significantly elevated when apigenin was applied with MK silencing. Combined treatment of CD44+CD133+ PC3 cells with apigenin and MK siRNA was also more effective in inducing apoptotic and non-apoptotic cell death when compared with individual applications. Treatment of CD44+ LNCaP cells with apigenin significantly decreased viability, although the combination treatment did not markedly alter the individual therapy. Molecular events underlying cell cycle arrest and inhibition of the survival, proliferation, and migration of CD44+CD133+ PC3 cells were found to be associated with upregulated p21, p27, Bax, Bid, caspase-3, and caspase-8 expression, as well as downregulated p-p38, p-ERK, NF-κB, and PARP. In addition, the combination of apigenin treatment and MK silencing showed better outcomes on the anticancer efficacy of docetaxel in CD44+CD133+ PC3 cells. In conclusion, MK-regulated events are different between PCSCs, and when combined with apigenin plus MK silencing, docetaxel treatment may be a valuable approach for the eradication of PCSCs.

Midkine downregulation increases the efficacy of quercetin on prostate cancer stem cell survival and migration through PI3K/AKT and MAPK/ERK pathway.

AIMS: To examine the functions of growth factor midkine (MK) and a flavonoid quercetin on survival, apoptosis and migration of prostate cancer (PCa) stem cells (CSCs). MAIN METHODS: CD44+/CD133+ and CD44+ stem cells were isolated from PC3 and LNCaP cells, respectively by magnetic-activated cell sorting system. 3D cell culture was used to evaluate the ability of quercetin, MK siRNA, and the combination of both to inhibit spheroid formation, apoptosis and cell cycle arrest. Image-based cytometer, RT-qPCR, Western blotting and transwell migration assays were performed. KEY FINDINGS: Quercetin treatment for 24-72 h inhibited PC3 and CD44+/CD133+ stem cell proliferation in a time- and dose-dependent manner. Knockdown of endogenous MK expression significantly suppressed proliferation of CD44+/CD133+ and CD44+ cells as well as their parent cells. Co-administration of MK siRNA and quercetin reduced the cell survival, induced apoptosis and caused G1 phase cell cycle arrest more effectively than the individual therapy. Knockdown of MK significantly enhanced the inhibitory effect of quercetin on CD44+/CD133+ migration and spheroid formation. In addition, the combined therapy inhibited the phosphorylation of PI3K, AKT and ERK1/2, and reduced the protein expression of p38, ABCG2 and NF-κB. SIGNIFICANCE: Quercetin alone exhibited significant cytotoxic effects on CD44+/CD133+. MK plays an important role in the proliferation of CD44+/CD133+ and CD44+ cells in particular, and quercetin and MK-silencing therapy may be an important strategy in targeting CSCs that play a role in relapse, migration and drug resistance.

The flavonoid apigenin reduces prostate cancer CD44(+) stem cell survival and migration through PI3K/Akt/NF-κB signaling.

AIMS: Cancer stem cells (CSCs) are involved in drug resistance, metastasis and recurrence of cancers. The efficacy of apigenin on cell survival, apoptosis, migration and stemness properties were analyzed in CSCs. MAIN METHODS: Prostate CSCs (CD44(+)) were isolated from human prostate cancer (PCa) PC3 cells using a magnetic-activated cell sorting system. PC3 and CSCs were treated with various concentrations of apigenin, docetaxel and their combinations for 48h. KEY FINDINGS: Apigenin dose dependently inhibited CSCs and PC3 cell survival, and this was accompanied with a significant increase of p21 and p27. Apigenin induced apoptosis via an extrinsic caspase-dependent pathway by upregulating the mRNA expressions of caspases-8, -3 and TNF-α, but failed to regulate the intrinsic pathway as determined by the Bax, cytochrome c (Cyt-c) and APAF-1 in CSCs. In contrary to CSCs, apigenin induced intrinsic apoptosis pathway as evidenced by the induction of Bax, Cyt-c and caspase-3 while caspase-8, TNF-α and Bcl-2 levels remained unchanged in PC3 cells. The flavonoid strongly suppressed the migration rate of CSCs compared to untreated cells. Significant downregulation of matrix metallopeptidases-2, -9, Snail and Slug exhibits the ability of apigenin treatment to suppress invasion. The expressions of NF-κB p105/p50, PI3K, Akt and the phosphorylation of pAkt were decreased after apigenin treatment. Moreover, apigenin treatment significantly reduced pluripotency marker Oct3/4 protein expression which might be associated with the down-regulation of PI3K/Akt/NF-κB signaling. SIGNIFICANCE: Our data indicated that, apigenin could be a useful compound to prevent proliferation and migration of cancer cells as well as CSCs.

Rational design of an immunoconjugate for selective knock-down of leukemia-specific E2A-PBX1 fusion gene expression in human Pre-B leukemia.

The t(1;19)(q23;p13) is one of the most common chromosomal translocations in acute lymphoblastic leukemia (ALL) and results in production of the transforming oncoprotein E2A-PBX1. Here we first report a novel, biomarker-guided biotherapy strategy for personalized treatment of t(1;19)(+) ALL. A supervised interrogation of the gene expression profiles of primary leukemic cells from a cohort of 207 children with high risk B-lineage ALL identified up-regulated CD19 gene expression as a biomarker for t(1;19)(+) ALL. A disulfide-linked immunoconjugate of a 5-amino-modified 24 mer phosphorothioate anti-sense E2A-PBX1 oligonucleotide (AON) with a mAb specific for a CD19 receptor (αCD19-AON) was prepared as a CD19-directed and leukemia-specific biotherapeutic agent against E2A-PBX1(+) B-lineage ALL. Treatment of E2A-PBX1(+) leukemia cells with low nanomolar concentrations of αCD19-AON resulted in selective depletion of E2A-PBX1 transcripts and caused apoptotic destruction and abrogation of clonogenic growth. Subcutaneously administered αCD19-AON at a total dose level of 93 nmol kg(-1) delivered over 14 days using a micro-osmotic pump more than doubled the leukemia-free survival time of SCID mice in a xenograft model of E2A-PBX1(+) human B-lineage ALL (82.0 ± 1.9 days vs. 37.0 ± 0.1 days, P < 0.0001). Both the AON moiety and the targeting CD19-specific mAb moiety were required for the in vitro as well as in vivo anti-leukemic activity of αCD19-AON. The observed in vitro and in vivo anti-leukemic potency of the αCD19-AON immunoconjugate provides the first preclinical proof-of-principle that t(1;19)(+) high risk B-lineage ALL can be treated with leukemia-specific biotherapeutic agents that knock-down E2A-PBX1 expression.

In vitro and in vivo chemosensitizing activity of LFM-A13, a dual-function inhibitor of Bruton's tyrosine kinase and polo-like kinases, against human leukemic B-cell precursors.

The present study documents the chemosensitizing anti-leukemic activity of the leflunomide metabolite (LFM) analog, LFM-A13, a dual-function inhibitor of Bruton's tyrosine kinase (BTK) and Polo-like kinases (PLK), against human leukemic B-cell precursors. The results in 135 xenografted NOD/SCID mice regarding the anti-leukemic activity of GMP-grade LFM-A13, obtained with only 4-days of LFM-A13 therapy at nontoxic dose levels corresponding to 1-20% of its NOAEL (no observable advserse effect level), alone or in combination with the standard chemotherapy drug vincristine, demonstrate the potential of LFM-A13 as a new anti-leukemic drug candidate. All 82 LFM-A13-treated mice, including those receiving a combination of vincristine + LFM-A13 at the highest dose level of LFM-A13, tolerated their treatments well without weight loss, diarrhea, lethargy/ paralysis, other signs of morbidity, or mortality. The present study provides preclinical proof-of-principle for the development of LFM-A13 as a new chemosensitizing and apoptosis-promoting anti-leukemic agent and lends support to the hypothesis that the chemoresistance of relapsed B-cell precursor acute lymphoblastic leukemia (BCP-ALL) can be overcome by using LFM-A13 in combination with chemotherapy. Also presented are the results of a comprehensive meta-analysis of the overexpression of genes for LFM-A13 targeted kinases and their downstream effector molecules in B-lineage lymphoid malignancies utilizing the Oncomine database.

Chemoprevention of colorectal cancer by targeting Janus kinase 3 with a rationally designed small molecule inhibitor.

The effects of the rationally designed JAK3 inhibitor JANEX-1 on the development of intestinal tumors in the APC(min) mouse model of familial adenomatous polyposis were examined. At a non-toxic dose level, >4 times lower than its day 30 LD(10), JANEX-1 was highly effective in preventing intestinal tumor development in Min mice, resulting in markedly improved survival outcomes. JAK3 inhibitors may, therefore, be useful in the chemoprevention of colorectal cancer. Here, an overview regarding the potential of JANEX-1 as a chemopreventive agent is provided.

CD22 EXON 12 deletion as a pathogenic mechanism of human B-precursor leukemia.

Here, we report that primary leukemic cells from infants with newly diagnosed B-precursor leukemia express a truncated and functionally defective CD22 coreceptor protein that is unable to transmit apoptotic signals because it lacks most of the intracellular domain, including the key regulatory signal transduction elements and all of the cytoplasmic tyrosine residues. Expression of this structurally and functionally abnormal CD22 protein is associated with a very aggressive in vivo growth of patients' primary leukemia cells causing disseminated overt leukemia in SCID mice. The abnormal CD22 coreceptor is encoded by a profoundly aberrant mRNA arising from a splicing defect that causes the deletion of exon 12 (c.2208-c.2327) (CD22ΔE12) and results in a truncating frameshift mutation. The splicing defect is associated with multiple homozygous mutations within a 132-bp segment of the intronic sequence between exons 12 and 13. These mutations cause marked changes in the predicted secondary structures of the mutant CD22 pre-mRNA sequences that affect the target motifs for the splicing factors hnRNP-L, PTB, and PCBP that are up-regulated in infant leukemia cells. Forced expression of the mutant CD22ΔE12 protein in transgenic mice perturbs B-cell development, as evidenced by B-precursor/B-cell hyperplasia, and corrupts the regulation of gene expression, causing reduced expression levels of several genes with a tumor suppressor function. We further show that CD22ΔE12-associated unique gene expression signature is a discriminating feature of newly diagnosed infant leukemia patients. These striking findings implicate CD22ΔE12 as a previously undescribed pathogenic mechanism in human B-precursor leukemia.

Prevention of UVB-induced skin inflammation, genotoxicity, and photocarcinogenesis in mice by WHI-P131, a dual-function inhibitor of Janus kinase 3 and EGF receptor kinase.

The current study examined the chemopreventive potential of the dual-function JAK3/EGFR tyrosine kinase inhibitor WHI-P131 (CAS 202475-60-3) in photocarcinogenesis of non-melanoma skin cancer (NMSC). Prophylactic WHI-P131 exhibited significant anti-inflammatory activity in the SKH-1 mouse model of sunburn and afforded significant protection against the inflammatory skin damage that results from UVB exposure. UVB exposure (400 mJ/cm2) increased the mutation rate of the transgene target in UVB-exposed skin of BigBlue mice by a factor of 3.7 from 8.6 x 10(-5) to 31.7 x 10(-5) but this genotoxicity was almost completely prevented by topically administered prophylactic WHI-Pl31 (1.5 mg/cm2). Chronic and repetitive exposure of vehicle-treated SKH-1 mice to 35 mJ/cm2 UVB, three times per week for 20 weeks resulted in appearance of a spectrum of lesions from actinic keratoses and squamous cell carcinoma (SCC) in situ to invasive SCC. Both the number and size of the skin lesions progressively increased over time. Notably, topical administration of WHI-P131 (1.0 mg/cm2) over the UVB target skin area on the dorsal surface 15 min before each UVB exposure significantly suppressed the photocarcinogenesis as documented by a 4-week delay in the onset of visible skin lesions, decreased total lesion volume per mouse (1.9 +/- 0.5 mm3 vs. 2.5 +/- 0.5 mm3/lesion at 20 weeks), and decreased number (1.6 +/- 0.4/mouse vs. 4.2 +/- 1.6/mouse at 20 weeks, P < 0.05) as well as smaller size of lesions and consequently a smaller total lesion volume ("skin cancer burden") (10.6 +/- 4.3 mm3 vs. 3.2 +/- 0.9 mm3 at 20 weeks, P < 0.05). These experimental findings provide unprecedented evidence that WHI-P131 may be useful as a chemopreventive agent against NMSC.

Therapeutic nanoparticle constructs of a JAK3 tyrosine kinase inhibitor against human B-lineage ALL cells.

WHI-P131 (CAS 202475-60-3) is a dual-function inhibitor of JAK3 tyrosine kinase that demonstrated potent in vivo anti-inflammatory and anti-leukemic activity in several preclinical animal models. This is the first report of the development of nanoparticle (NP) constructs ofWHI-P131. Fourty-eight distinct NP formulations were prepared and WHI-P131 encapsulation efficiencies > 95% and intraliposomal WHI-P131 concentrations >10 mg/mL were achieved in lead NP formulations. The anti-cancer activity of WHI-P131-NP, a PEGylated lead formulation was tested in vitro and in vivo. Notably, WHI-P131-NP was capable of causing apoptotic death in primary leukemia cells from chemotherapy-resistant acute lymphoblastic leukemia (ALL) as well as chronic lymphocytic leukemia (CLL) patients. WHI-P131-NP was also active in the RS4;11 SCID mouse xenograft model of chemotherapy-resistant B-lineage ALL. The life table analysis showed that WHI-P131-NP was more effective than WHI-P131 (P = 0.01), vincristine (P < 0.0001), or vehicle (P < 0.0001). These experimental results demonstrate that the nanotechnology-enabled delivery of WHI-P131 shows therapeutic potential against leukemias with constitutive activation of the JAK3-STAT3/STAT5 molecular target.

Targeting JAK3 tyrosine kinase-linked signal transduction pathways with rationally-designed inhibitors.

Inhibitors of Janus Kinase 3 (JAK3) show potential as a new class of apoptosis-inducing anti-cancer drugs. In addition, JAK3 inhibitors may also be useful as immunosuppressive agents. Rationally designed selective inhibitors of JAK3 such as JANEX-1, that do not inhibit other Janus kinases have recently undergone extensive preclinical testing that revealed a favorable pharmacodynamic profile. Here we discuss the clinical potential of targeting JAK3-linked signal transduction pathways with small molecule inhibitors such as JANEX-1.

Anti-breast cancer activity of LFM-A13, a potent inhibitor of Polo-like kinase (PLK).

Molecular modeling studies led to the identification of LFM-A13 (alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide) as a potent inhibitor of Polo-like kinase (Plk). LFM-A13 inhibited recombinant purified Plx1, the Xenopus homolog of Plk, in a concentration-dependent fashion, as measured by autophosphorylation and phosphorylation of a substrate Cdc25 peptide. LFM-A13 was a selective Plk inhibitor. While the human PLK3 kinase was also inhibited by LFM-A13 with an IC(50) value of 61 microM, none of the 7 other serine/threonine kinases, including CDK1, CDK2, CDK3, CHK1, IKK, MAPK1 or SAPK2a, none of the 10 tyrosine kinases, including ABL, BRK, BMX, c-KIT, FYN, IGF1R, PDGFR, JAK2, MET, or YES, or the lipid kinase PI3Kgamma were inhibited (IC(50) values >200-500 microM). The mode of Plk3 inhibition by LFM-A13 was competitive with respect to ATP with a K(i) value of 7.2 microM from Dixon plots. LFM-A13 blocked the cell division in a zebrafish (ZF) embryo model at the 16-cell stage of the embryonic development followed by total cell fusion and lysis. LFM-A13 prevented bipolar mitotic spindle assembly in human breast cancer cells and glioblastoma cells and when microinjected into living epithelial cells at the prometaphase stage of cell division, it caused a total mitotic arrest. Notably, LFM-A13-delayed tumor progression in the MMTV/neu transgenic mouse model of HER2 positive breast cancer at least as effectively as paclitaxel and gemcitabine. LFM-A13 showed a favorable toxicity profile in mice and rats. In particular there was no evidence of hematologic toxicity as documented by peripheral blood counts and bone marrow examinations. These results establish LFM-A13 as a small molecule inhibitor of Plk with in vitro and in vivo anti-proliferative activity against human breast cancer.

Anti-cancer activity profile of 3'-azidothymidine 5'-[p-methoxyphenyl methoxyalaninyl phosphate] (Compound 003), a novel nucleoside analog.

The novel cytotoxic nucleoside analog Compound 003 (3'-azidothymidine 5'-[p-methoxyphenyl methoxyalaninyl phosphate], CAS 149560-32-7) prevented bipolar mitotic spindle assembly and caused a G2 arrest in human cancer cells. Compound 003 was very well tolerated by both mice and rats without any toxicity at cumulative dose levels >2 g/kg. Notably, Compound 003 prolonged cancer-free survival in the MMTVneu transgenic mouse model of HER2 positive breast cancer. These results indicate that Compound 003 may be useful in the treatment of cancer patients.

In vitro and in vivo pharmacokinetic features and metabolism of the novel cytotoxic nucleoside analog 3'-azidothymidine 5'-[p-methoxyphenyl methoxyalaninyl phosphate] (Compound 003).

The pharmacokinetic features and metabolism of the novel cytotoxic nucleoside analog Compound 003 (3'-azidothymidine 5'-[p-methoxyphenyl methoxyalaninyl phosphate], CAS 149560-32-7) were studied in both human cancer cells and mice. In mice, Compound 003 was rapidly converted into ala-AZT-MP (CAS 209214-06-2) and zidovudine (azidothymidine, AZT, CAS 30516-87-1). Maximum ala-AZT-MP concentrations were reached almost immediately (tmax < 5 min), while 50.4 min and 143.5 min were required to reach maximum AZT concentrations after intravenous and oral administration, respectively. The results indicate that paraoxon-sensitive carboxylesterases play an important role in the conversion of Compound 003 to ala-AZT-MP. This study provides the basis for future preclinical as well as clinical pharmacodynamic studies of Compound 003.