Cytarabine

Synthetic Routes and Reaction Conditions

Cytarabine is synthesized from cytidine through a series of chemical reactions. The traditional preparation method involves the use of polyphosphoric acid to synthesize this compound from cytidine. The reaction conditions typically include heating and the use of solvents such as water and alcohol. The yield of this process ranges from 29% to 40% .

Industrial Production Methods

In industrial settings, this compound is produced in large quantities using similar synthetic routes but with optimized reaction conditions to maximize yield and purity. The process involves the use of advanced purification techniques to ensure the final product meets pharmaceutical standards .

Hydrolysis and Deamination

Cytarabine undergoes pH-dependent hydrolysis and enzymatic deamination to form 1-β-D-arabinofuranosyluracil (ara-U) , its primary inactive metabolite.

Key Reactions:

• Deamination by cytidine deaminase :
  This compound → ara-U (via NH₃ elimination)
  • Activation energy : 28.24 kJ/mol (in citric acid) vs. 23.21 kJ/mol (in saline)
• Acid-catalyzed hydrolysis :
  Degrades via cleavage of the glycosidic bond at pH < 3 .
• Base-catalyzed degradation :
  Forms uracil arabinoside under alkaline conditions (pH > 9) .

Table 1: Thermodynamic Parameters of this compound Degradation

Phosphorylation and DNA Incorporation

This compound requires intracellular activation via phosphorylation to exert cytotoxic effects:

Metabolic Pathway:

• Phosphorylation by deoxycytidine kinase → This compound monophosphate (ara-CMP)
• Subsequent phosphorylation → This compound triphosphate (ara-CTP)
• Incorporation into DNA :
  • Competes with deoxycytidine triphosphate (dCTP) during DNA synthesis
  • Causes chain termination and destabilizes DNA duplexes (e.g., Okazaki fragments)

Table 2: Kinetic Effects of this compound on Model Okazaki Fragments

Interaction with Enzymatic Targets

This compound’s reactivity is governed by its inhibition of key enzymes:

Enzymatic Inhibition:

• DNA polymerase α/β : Direct competitive inhibition (Kᵢ = 0.1–1.0 μM)
• Ribonucleotide reductase : Reduces dCTP pools, enhancing ara-CTP incorporation
• Cytidine deaminase : Primary catabolic enzyme (Km = 0.2 mM)

Table 3: Stability in Clinical Formulations

Photodegradation and Oxidative Pathways

Advanced oxidation processes (AOPs) degrade this compound in wastewater:

• UV/H₂O₂ systems : Achieve >95% degradation via hydroxyl radical (·OH) attack
• Fenton-like reactions : Fe³⁺/UV systems yield 80% degradation in 60 min
• Primary degradation products : Arabinofuranosyluracil, cytosine, and arabinose

Solid-State Reactivity

This compound exhibits distinct hydrogen-bonding patterns influencing stability:

• Intramolecular H-bonds : O2’–H···O5’ stabilizes arabinose conformation
• Crystal packing : NH···O interactions with Gln97/Asp133 in dCK complexes

Compatibility with Excipients

This compound demonstrates variable stability in parenteral admixtures:

Pharmacokinetics

Cytarabine is poorly absorbed when taken orally due to extensive first-pass metabolism; therefore, it is typically administered via intravenous or subcutaneous routes. Its pharmacokinetic properties include:

• Bioavailability: High when administered intravenously.
• Volume of Distribution: High due to low plasma protein binding.
• Metabolism: Primarily occurs in the liver with renal excretion of metabolites .

Clinical Applications

This compound has a wide range of applications in oncology, particularly for treating different types of leukemia. Below are some key indications:

Liposomal Formulations

Recent advancements have led to the development of liposomal formulations of this compound, which improve its pharmacokinetic profile and reduce systemic toxicity. Liposomal this compound has shown efficacy in treating CNS relapses in ALL and has been compared favorably to traditional therapies .

Efficacy Studies

A study evaluating liposomal this compound for CNS relapse in ALL demonstrated comparable safety and efficacy profiles to methotrexate and traditional this compound formulations. The European Working Group for Adult ALL initiated trials to further explore this application .

Case Studies

• Case Study: Liposomal this compound in CNS Relapse
  • Patient Profile: Adult patient with recurrent ALL.
  • Treatment Regimen: Administered liposomal this compound.
  • Outcome: Achieved significant CNS response after one cycle; tolerated well without severe adverse effects.
• Case Study: Differentiation Induction
  • Patient Profile: AML patient receiving low-dose this compound.
  • Treatment Regimen: Low-dose this compound combined with differentiation agents.
  • Outcome: Induced maturation of leukemic cells without significant toxicity, suggesting potential as a differentiation therapy.

Cytarabine exerts its effects by inhibiting DNA polymerase, an enzyme crucial for DNA synthesis. It is incorporated into DNA during the S-phase of the cell cycle, leading to the termination of DNA chain elongation and ultimately causing cell death. This mechanism makes this compound particularly effective against rapidly dividing cancer cells .

Similar Compounds

Fludarabine: Another nucleoside analog used in the treatment of leukemia.

Cladribine: Used to treat hairy cell leukemia and multiple sclerosis.

Gemcitabine: Employed in the treatment of various cancers, including pancreatic and breast cancer.

Uniqueness of Cytarabine

This compound is unique due to its specific mechanism of action, targeting DNA polymerase and its incorporation into DNA. This specificity makes it highly effective in treating certain types of leukemia. Additionally, its use in liposomal formulations has shown reduced cardiotoxicity compared to other chemotherapeutic agents .

Cytarabine, also known as cytosine β-D-arabinofuranoside, is a pyrimidine nucleoside analog primarily used in the treatment of acute myeloid leukemia (AML) and other hematological malignancies. Its biological activity is characterized by its ability to inhibit DNA synthesis, thereby exerting cytotoxic effects on rapidly dividing cells. This article explores the mechanisms of action, pharmacokinetics, clinical efficacy, and recent advancements in drug formulations related to this compound.

This compound's primary mechanism involves its conversion into this compound triphosphate (Ara-CTP) within the cell. This active form competes with deoxycytidine triphosphate (dCTP) for incorporation into DNA during replication. The incorporation of Ara-CTP results in the termination of DNA chain elongation, leading to cell cycle arrest at the S-phase and subsequent apoptosis of malignant cells.

• Key Enzymes Involved :
  • Deoxycytidine kinase (DCK) activates this compound by phosphorylating it to Ara-CTP.
  • Cytidine deaminase (CDA) is responsible for the catabolism of this compound, influencing its bioavailability and efficacy.

Pharmacokinetics

This compound has a short plasma half-life, typically around 1 to 3 hours, necessitating continuous infusion or frequent dosing to maintain therapeutic levels. Its pharmacokinetic profile can be affected by various factors including patient genetics and concurrent medications.

Clinical Efficacy

This compound is a cornerstone in the treatment regimens for AML and is often used in combination with other agents such as anthracyclines. The efficacy varies among patients due to genetic polymorphisms affecting drug metabolism.

Case Studies

• Ewing Sarcoma : A Phase II study demonstrated that intermediate doses of this compound significantly reduced EWS/FLI1 protein levels in Ewing sarcoma cells both in vitro and in vivo, indicating potential utility beyond traditional leukemias .
• Acute Myeloid Leukemia : Clinical trials have shown that high-dose this compound regimens improve remission rates in patients with AML, particularly those with favorable cytogenetics .

Recent Advancements

Recent research has focused on enhancing the delivery and efficacy of this compound through novel drug formulations:

• Cholic Acid-Cytarabine Conjugates : These liver-targeting conjugates have shown improved absorption and specificity for hepatic tissues, potentially reducing systemic toxicity while maintaining antitumor activity .
• Pharmacogenomic Biomarkers : Studies are exploring genetic markers that predict patient response to this compound therapy, aiming to tailor treatments based on individual metabolic profiles .

Basic Research Questions

Q. What experimental methodologies are recommended for elucidating cytarabine’s mechanism of action in acute myeloid leukemia (AML) cells?

• Methodological Answer : Use in vitro assays to measure this compound’s incorporation into DNA via competitive inhibition of DNA polymerase. Employ metabolomic profiling (e.g., LC-MS) to track the activation of this compound by deoxycytidine kinase and its deactivation by cytidine deaminase . Combine flow cytometry with Annexin V/PI staining to quantify apoptosis in AML cell lines (e.g., HL-60) after this compound exposure. For in vivo models, utilize patient-derived xenografts (PDX) to correlate drug efficacy with tumor regression .

Q. How can researchers standardize cytotoxicity assays to ensure reproducibility in this compound studies?

• Methodological Answer : Adopt the MTT or CellTiter-Glo assays for viability measurements, with strict controls for cell density, incubation time, and this compound concentration gradients (e.g., 0.1–10 μM). Include positive controls (e.g., daunorubicin) and negative controls (untreated cells). Validate results across multiple cell lines (e.g., THP-1, MOLM-13) and replicate experiments ≥3 times. Document all protocols using FAIR data principles .

Q. What analytical techniques are most effective for quantifying intracellular this compound metabolites?

• Methodological Answer : Use high-performance liquid chromatography (HPLC) with UV detection or liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure this compound triphosphate (Ara-CTP) levels. Optimize sample preparation to prevent metabolite degradation (e.g., rapid freezing in liquid nitrogen). Cross-validate results with enzymatic assays targeting deoxycytidine kinase activity .

Advanced Research Questions

Q. How do genetic mutations (e.g., TP53, FLT3-ITD) influence this compound resistance in AML, and what experimental models best recapitulate this?

• Methodological Answer : Perform whole-exome sequencing on this compound-resistant AML samples to identify co-occurring mutations. Use CRISPR-Cas9 to introduce mutations (e.g., TP53 KO) into isogenic cell lines and assess IC50 shifts. Validate findings in PDX models treated with this compound monotherapy. Analyze RNA-seq data to identify dysregulated pathways (e.g., DNA repair, nucleoside transport) .

Q. What strategies optimize this compound’s pharmacokinetic profile in preclinical models to enhance blood-brain barrier penetration?

• Methodological Answer : Develop liposomal or nanoparticle formulations to improve this compound’s stability and bioavailability. Use pharmacokinetic modeling (e.g., non-compartmental analysis) to calculate AUC and half-life in murine models. Validate CNS delivery via cerebrospinal fluid (CSF) sampling and compare tumor burden in brain xenografts vs. systemic models .

Q. How can researchers resolve contradictory data on this compound’s synergy with anthracyclines (e.g., daunorubicin) across different AML subtypes?

• Methodological Answer : Conduct dose-matrix experiments (e.g., Chou-Talalay synergy assays) to calculate combination indices (CI) in genetically annotated AML cell lines. Stratify results by molecular subgroups (e.g., NPM1 mutant vs. wild-type). Use RNA interference to knock down putative resistance genes (e.g., ABCG2) and reassess synergy .

Q. What epigenetic biomarkers (e.g., DNA methylation, miRNA) predict this compound response, and how can they be validated clinically?

• Methodological Answer : Perform reduced-representation bisulfite sequencing (RRBS) or methylome arrays on pretreatment AML biopsies. Correlate methylation patterns (e.g., CDKN2B hypermethylation) with complete remission rates. Validate candidates in prospective cohorts using droplet digital PCR (ddPCR) for targeted methylation analysis .

Q. How should researchers design studies to evaluate this compound’s off-target effects on normal hematopoietic stem cells (HSCs)?

• Methodological Answer : Use colony-forming unit (CFU) assays to compare this compound’s impact on CD34+ HSCs vs. AML blasts. Employ single-cell RNA-seq to identify HSC subpopulations with differential sensitivity. Validate findings in ex vivo cultures of healthy donor-derived HSCs .

Q. Guidance for Addressing Methodological Challenges

• Data Contradictions : Apply Bradford Hill criteria to assess causality in conflicting studies. For example, if this compound shows efficacy in in vitro models but fails in PDX, evaluate differences in tumor microenvironment (e.g., stromal cell interactions) using spatial transcriptomics .
• Translational Gaps : Use patient-derived organoids (PDOs) to bridge in vitro and clinical data. Test this compound combinations in PDOs and correlate results with matched patient outcomes .