Cyclophosphamide

Pharmacokinetics of Cyclophosphamide and its Metabolites

This compound is a prodrug that requires metabolic activation, primarily in the liver, to exert its effects. frontiersin.orgnih.govnih.gov The pharmacokinetic profile of CP involves its absorption, distribution, metabolism into active and inactive metabolites, and subsequent elimination. nih.govdrugbank.com

Pharmacodynamic Considerations in Research

Pharmacodynamic research related to this compound focuses on understanding how the drug and its active metabolites interact with biological systems to produce their effects. The primary mechanism of action of this compound's active metabolite, phosphoramide mustard, involves the formation of DNA cross-links. nih.govwikipedia.org These cross-links occur both between and within DNA strands, primarily at the N-7 positions of guanine bases. wikipedia.org This DNA damage interferes with DNA synthesis and RNA transcription, ultimately leading to cell apoptosis. wikipedia.org

Research explores the relationship between this compound exposure (often measured by area under the concentration-time curve - AUC) and pharmacodynamic endpoints. Studies have investigated the correlation between this compound or metabolite exposure and biological responses. frontiersin.orgresearchgate.netaacrjournals.org For instance, research has explored the association between this compound exposure (AUC) and neutropenic toxicity in clinical studies, indicating a positive correlation. frontiersin.org

Furthermore, research explores the influence of genetic factors and drug interactions on the pharmacodynamic effects of this compound by altering its metabolism and the subsequent exposure to active metabolites. aacrjournals.orgresearchgate.netnih.gov Studies investigate how variations in enzyme activity or the presence of other compounds can impact the levels of active metabolites and, consequently, the pharmacodynamic response. aacrjournals.orgnih.gov

Genetic Polymorphisms of Cytochrome P450 Enzymes

Cyclophosphamide is primarily activated by hepatic CYP450 enzymes through 4-hydroxylation, leading to the formation of 4-hydroxythis compound, which is in equilibrium with aldophosphamide. frontiersin.orgpharmgkb.orgresearchgate.net This activation is mediated by several CYP isoforms, with CYP2B6, CYP2C9, CYP2C19, and CYP3A4 playing significant roles, while CYP2A6 and CYP2C8 make more minor contributions. frontiersin.orgpharmgkb.orgfrontiersin.orgresearchgate.net Genetic polymorphisms within the genes encoding these enzymes can lead to altered enzyme activity and expression, contributing to variability in this compound metabolism. frontiersin.orgfrontiersin.orgnih.gov

Polymorphisms in Detoxification Enzymes (e.g., Glutathione S-transferases, ALDH)

Detoxification of this compound metabolites is crucial for reducing toxicity. This process primarily involves phase II inactivation enzymes, including aldehyde dehydrogenases (ALDHs) and glutathione S-transferases (GSTs). tandfonline.comfrontiersin.orgpharmgkb.org

Aldehyde Dehydrogenases (ALDHs): ALDH enzymes, particularly ALDH1A1 and ALDH3A1, play a major role in oxidizing aldophosphamide to the inactive metabolite carboxyphosphamide. frontiersin.orgpharmgkb.orgnih.govucsc.eduwikipedia.orgresearchgate.net Higher levels of ALDH activity in cells, such as hematopoietic stem cells, can confer resistance to this compound. frontiersin.orgucsc.edu Polymorphisms in ALDH genes can affect enzyme activity and thus influence the rate of detoxification. tandfonline.comnih.govnih.gov For example, the ALDH1A1 rs8187996 variant has been explored for its association with this compound toxicity, although findings have been conflicting. nih.govpharmgkb.org

Glutathione S-transferases (GSTs): GSTs, including GSTA1, GSTM1, GSTP1, and GSTT1, are involved in conjugating this compound metabolites, such as acrolein and phosphoramide mustard, with glutathione (GSH), facilitating their detoxification and excretion. tandfonline.comfrontiersin.orgpharmgkb.orgmdpi.comnih.govashpublications.orgmdpi.com Polymorphisms in GST genes can lead to decreased detoxification capacity. nih.govfrontiersin.orgmdpi.com The GSTM1-null genotype and GSTA1*B haplotype have been identified as genetic factors contributing to the risk of sinusoidal obstruction syndrome (SOS). frontiersin.orgashpublications.org Polymorphisms in GSTP1 that reduce its ability to detoxify this compound and its metabolites may be associated with superior survival outcomes but also an increased risk of toxicity. nih.govtandfonline.com For instance, the GSTP1 p.Ile105Val (rs1695) polymorphism has been analyzed in relation to this compound toxicity, with some studies suggesting that the Val/Val genotype may be associated with an increased risk of myelosuppression and gastrointestinal toxicity. oncotarget.com

Polymorphisms in detoxification genes that result in lowered enzyme activity can lead to decreased elimination of this compound metabolites, potentially increasing the levels of toxic metabolites. nih.govfrontiersin.org

Impact of Genetic Variants on this compound Activation and Inactivation

Genetic variants in both activation (CYP) and inactivation (ALDH, GST) enzymes significantly impact the balance between the formation of active cytotoxic metabolites and inactive detoxification products. tandfonline.comnih.govnih.govnih.govhznu.edu.cn Polymorphisms in CYP enzymes that lead to reduced activity can result in lower production of the active metabolite 4-hydroxythis compound/aldophosphamide. nih.govfrontiersin.orgresearchgate.netmdpi.com Conversely, polymorphisms in detoxification enzymes that impair their function can lead to an accumulation of toxic metabolites like phosphoramide mustard and acrolein. nih.govfrontiersin.orgmdpi.com

Pharmacogenetic Associations with Efficacy and Toxicity Outcomes

Numerous studies have investigated the associations between genetic polymorphisms in this compound metabolizing enzymes and clinical outcomes, including efficacy and toxicity. tandfonline.comnih.govnih.govnih.govmdpi.comhznu.edu.cn Genetic factors are recognized as playing a role in the interindividual variability observed in response and toxicities associated with this compound-based therapies. nih.govtandfonline.compharmgkb.orgresearchgate.net

Observed associations include:

Polymorphisms in CYP2B6, a major activation enzyme, have been associated with decreased activity and a higher risk of graft-versus-host disease (GVHD). frontiersin.org

Variants in other activation enzymes (CYP2A6, CYP2C8, CYP2C9, CYP2C19) leading to decreased enzyme activity have also been associated with GVHD. frontiersin.orgresearchgate.net

Polymorphisms in detoxification genes like glutathione S-transferases, which decrease the ability to detoxify metabolites, have been linked to increased amounts of toxic metabolites and the development of severe acute GVHD. nih.govfrontiersin.org

The GSTM1*0 polymorphism has been recognized as a risk factor for sinusoidal obstruction syndrome (SOS). nih.govfrontiersin.org

Polymorphisms that reduce the detoxification capacity of GSTP1 may be associated with both superior survival outcomes and an increased risk of toxicity, including secondary malignancies. nih.govtandfonline.com

Studies have explored the association between CYP3A5 polymorphisms and this compound efficacy and toxicity, with some findings suggesting an association between CYP3A53 and CYP3A56 variants and outcomes in pediatric rhabdomyosarcoma patients. waocp.org

The CYP2C19 genotype has been reported to influence this compound pharmacokinetics, such as the half-life of the parent drug. nih.gov Reduced CYP2C19 activity may impair this compound activation, potentially reducing therapeutic efficacy. researchgate.netmdpi.com

While some studies have found associations between genetic variants and this compound toxicity, others have reported a lack of association for certain polymorphisms, such as CYP2B6. nih.govpharmgkb.org Conflicting data exists regarding the relationship between this compound efficacy, toxicity, and pharmacokinetics with genes involved in 4-hydroxythis compound formation and elimination. firstwordpharma.com Some research suggests that age and chemotherapy regimen, rather than germline pharmacogenomics, may be associated with this compound clearance or the ratio of 4-hydroxythis compound to this compound exposure in certain pediatric cancer patients. firstwordpharma.com

The complexity of this compound metabolism, involving multiple interacting enzymes and pathways, contributes to the challenge of definitively linking single genetic variants to clinical outcomes. researchgate.netresearchgate.net Further systematic and genome-wide pharmacogenomic studies are needed to verify current observations and identify additional genetic markers influencing this compound response and toxicity. nih.govtandfonline.com

Cardiotoxicity Mechanisms

Cardiotoxicity is a dose-limiting side effect of cyclophosphamide, typically observed at high doses. plos.orgekb.eg The precise mechanism is not fully established, but it is believed to involve oxidative stress and direct damage to endothelial capillaries. plos.orgnih.gov This damage can lead to the extravasation of proteins, erythrocytes, and toxic metabolites. nih.gov Breakdown of endothelial cells in the presence of toxic metabolites causes direct injury to the myocardium and capillary blood vessels, resulting in edema, interstitial hemorrhage, and the formation of microthrombi. nih.gov These insults can manifest clinically as acute heart failure and arrhythmias. nih.gov Histopathological findings in fatal cases have included increased cardiac weight, intramyocardial extravasation of blood, fibrin or fibrin-platelet microthrombi in capillaries, and fibrin in the interstitium. nih.gov Capillary microthrombosis and fibrin deposition in the myocardial interstitium have been noted as unique features of this compound-induced cardiac injury. nih.gov Electron microscopy has revealed features consistent with myocardial necrosis, such as hypercontraction bands, myofibrillar damage, lysis, intramitochondrial electron-dense inclusions, and fibrin deposition in myocyte cytoplasm. nih.gov this compound and its metabolites, including acrolein, 4-hydroxythis compound, and aldophosphamide, are identified as cardiotoxic agents, with acrolein considered particularly toxic to cardiomyocytes. ekb.eg Acrolein, an unsaturated highly reactive aldehyde, can damage cardiomyocytes, fibroblasts, and endothelial cells, leading to autophagy, inflammation, oxidative stress, and myocardial systolic dysfunction. ekb.egresearchgate.net this compound can induce oxidative stress in cardiac cells by reducing myocardial antioxidant concentrations. ekb.eg Increased intracellular Ca2+ concentration, potentially mediated by this compound, can contribute to pathological actions such as altered cardiac muscle contraction, inflammation of cardiac cells, and increased oxidative stress, leading to myocardial disorders like cardiomyopathy and heart failure. ekb.eg this compound intake has also been linked to an increase in nitric oxide, potentially through its effect on iNOS or eNOS. ekb.eg While eNOS can have a cardioprotective effect, its toxicity is mediated by the production of peroxynitrite (ONOO−), which causes inflammation, cell death, and cardiac toxicity. ekb.eg

Hemorrhagic Cystitis Pathogenesis (Acrolein-mediated)

Hemorrhagic cystitis is a significant adverse effect of this compound, primarily mediated by the urotoxic metabolite acrolein. medscape.commdpi.comnih.gov this compound is a prodrug that is metabolized in the liver by cytochrome P-450 enzymes to 4-hydroxythis compound, which is in equilibrium with aldophosphamide. plos.orgnih.gov Aldophosphamide can then decompose through β-elimination to form the cytotoxic phosphoramide mustard and acrolein. plos.orgecronicon.net Acrolein is excreted in the urine and accumulates in the bladder, where its prolonged contact with the urothelium causes damage. medscape.comeviq.org.au Acrolein is highly reactive and is considered the main molecule responsible for this side effect. nih.govecronicon.net The mechanism involves a complex and multimodal process leading to bladder edema and hemorrhage. medscape.comnih.gov Acrolein rapidly enters uroepithelial cells and activates intracellular reactive oxygen species and nitric oxide production, leading to peroxynitrite production. nih.gov This increased peroxynitrite level damages lipids (lipid peroxidation), proteins (protein oxidation), and DNA (strand breaks), resulting in the activation of poly(adenosine diphosphate-ribose) polymerase (PARP), a DNA repair enzyme. nih.gov Overactivation of PARP due to DNA damage leads to the depletion of oxidized nicotinamide-adenine dinucleotide and adenosine triphosphate, ultimately causing necrotic cell death. nih.gov Acrolein can perpetuate oxidative stress by inducing and bolstering lipid peroxidation and ROS generation. nih.gov Hemorrhagic cystitis is an inflammatory process, and cytokines such as tumor necrosis factor (TNF) and the interleukin (IL) family, as well as transcription factors like nuclear factor-kappaB (NF-κB) and activator protein-1 (AP-1), play a role in its pathogenesis. nih.gov Acrolein-induced hemorrhagic cystitis in mouse models causes apoptosis and necrosis of urothelial cells. nih.gov Inflamed bladder detrusor smooth muscle cells can also undergo autophagy following exposure to this compound or macrophage migration inhibitory factors. nih.gov

Neurotoxicity Mechanisms

This compound can induce neurotoxicity, which may manifest as anxiety, depression, motor dysfunction, and cognitive deficits. nih.gov Several molecular mechanisms are implicated in this compound-induced neurotoxicity, including oxidative stress, neuroinflammation, neuronal cell death, and neurotransmitter dysregulation. nih.govnih.gov

Myelosuppression and Immunosuppression Mechanisms

Myelosuppression and immunosuppression are significant toxicities associated with this compound. nih.govmedsafe.govt.nz These effects stem primarily from the action of phosphoramide mustard, the main active alkylating metabolite of this compound. wikipedia.orgnih.gov

This compound is converted by hepatic cytochrome P450 enzymes to 4-hydroxythis compound, which exists in equilibrium with aldophosphamide. wikipedia.orgpharmgkb.org Aldophosphamide can then decompose spontaneously or be enzymatically cleaved to form phosphoramide mustard and acrolein. pharmgkb.orgnih.govmims.com Phosphoramide mustard is the key cytotoxic metabolite responsible for the therapeutic effects of this compound, but it also targets rapidly proliferating cells in the bone marrow and immune system. wikipedia.orgnih.govxiahepublishing.com

Phosphoramide mustard exerts its effects by forming DNA crosslinks, both between and within DNA strands, primarily at the N-7 positions of guanine bases. wikipedia.orgnih.gov This irreversible DNA damage interferes with DNA replication and transcription, ultimately leading to cell cycle arrest and apoptosis in susceptible cells, including hematopoietic stem and progenitor cells in the bone marrow and various immune cells. wikipedia.orgxiahepublishing.com The suppression of hematopoietic stem cells and progenitor cells in the bone marrow is a characteristic feature of this compound-induced myelosuppression, leading to a decrease in blood cell counts, including leukocytes, erythrocytes, and platelets. medsafe.govt.nzfrontiersin.org

This compound can also induce oxidative stress in the bone marrow, which can contribute to myelosuppression by suppressing anti-oxidative enzymes. medrxiv.orgresearchgate.net Immunosuppression results from the depletion of lymphocytes, particularly T and B cells, due to the cytotoxic effects of phosphoramide mustard on these rapidly dividing immune cells. xiahepublishing.comfrontiersin.org This can lead to an increased susceptibility to infections. medsafe.govt.nz

Long-term Toxicities and Associated Mechanisms

Long-term this compound therapy is associated with several delayed toxicities, including an increased risk of secondary malignancies, infertility, and organ damage. nih.gov

The genotoxic nature of phosphoramide mustard, through its DNA alkylating activity and the induction of DNA damage, contributes to the increased risk of secondary cancers, particularly bladder cancer. nih.govbccancer.bc.ca While DNA repair mechanisms exist, persistent or unrepaired DNA lesions can lead to mutations and chromosomal aberrations, promoting malignant transformation over time.

Infertility is a significant long-term toxicity, particularly with higher cumulative doses of this compound. wikipedia.orgnih.govxiahepublishing.com The drug's metabolites, including phosphoramide mustard, directly damage germ cells in the ovaries and testes, which are also rapidly proliferating. xiahepublishing.com In females, this compound can cause primordial follicle loss, leading to premature ovarian insufficiency and amenorrhea. xiahepublishing.comnih.gov The mechanism involves DNA damage-induced apoptosis in oocytes and follicular cells. nih.gov Studies in mice have shown that this compound exposure can impair the developmental competence of oocytes through alterations in epigenetic modifications and the transcription of maternal factors. nih.gov In males, this compound can cause testicular damage and impair spermatogenesis in a dose-dependent manner. xiahepublishing.com

Organ toxicities, such as hemorrhagic cystitis, pulmonary fibrosis, and cardiotoxicity, can also manifest as long-term complications. nih.govbccancer.bc.ca Hemorrhagic cystitis is primarily attributed to the urotoxic effects of acrolein, another metabolite formed during the breakdown of aldophosphamide. pharmgkb.orgmims.combccancer.bc.ca Acrolein is an α,β-unsaturated aldehyde that is highly reactive and can cause direct injury to the urothelium. bccancer.bc.cawikipedia.org Pulmonary fibrosis, a less common but serious long-term effect, may involve direct injury to the pulmonary epithelium by this compound metabolites. bccancer.bc.ca Cardiotoxicity, while possible with high doses, does not appear to be cumulative in the same way as with anthracyclines but can still lead to long-term cardiac issues in some cases. nih.govbccancer.bc.ca The mechanism may involve direct injury to the endothelium by phosphoramide mustard. bccancer.bc.ca

While acrolein has historically been considered a major contributor to this compound toxicity, particularly urotoxicity, some research suggests that in vivo, the enzymatic decomposition of aldophosphamide primarily yields phosphoramide mustard and 3-hydroxypropanal (HPA), with acrolein formation being more prominent in in vitro settings. researchgate.netmdpi.com HPA is thought to amplify the apoptosis initiated by phosphoramide mustard's DNA alkylation. researchgate.netmdpi.com However, other sources still strongly associate acrolein with bladder toxicity. pharmgkb.orgmims.combccancer.bc.ca

Table: Key Metabolites and Associated Toxicities

In Vitro Models for Mechanistic Studies

In vitro models, primarily cell culture experiments, are essential for dissecting the cellular and molecular mechanisms of cyclophosphamide and its metabolites. These models allow for controlled investigations into the metabolic activation pathways, DNA alkylation, and downstream cellular responses such as apoptosis.

Studies using cell cultures have shown that phosphoreamide mustard (PAM), the active alkylating metabolite, is released from aldophosphamide (ALD) via β-elimination of acrolein in vitro. mdpi.com This differs from the in vivo process where ALD is cleaved by phosphodiesterases (PDE) to form PAM and 3-hydroxypropanal (HPA). mdpi.comecronicon.net The discovery of HPA as a CP metabolite and its proapoptotic properties, alongside the understanding that CP-induced cell death is p53-controlled apoptosis initiated by DNA alkylation, has contributed to the current understanding of CP's mechanism of action. mdpi.comecronicon.net

In vitro assays have also been used to investigate the direct effects of this compound and its metabolites on specific cell types. For instance, studies have explored the effects of CP on the differentiation and activity of osteoblasts and osteoclasts, revealing that CP can inhibit osteoblastogenesis by suppressing Wnt/β-catenin signaling and inhibit RANKL-mediated osteoclastogenesis by suppressing RANKL-activated signaling. oncotarget.com

Furthermore, in vitro cytotoxicity assays, such as the MTT assay, are commonly used to assess the effect of this compound and its formulations on cancer cells. mdpi.com These studies can also delve into the mechanism of cytotoxicity by analyzing the transcriptional levels of genes involved in apoptosis and cell cycle regulation, such as Caspase3, Caspase9, CyclinD, and CyclinE. mdpi.com

In Vivo Animal Models for Efficacy and Toxicity Research

In vivo animal models, particularly murine models, are indispensable for evaluating the efficacy and toxicity of this compound in a complex biological system before clinical trials. These models allow researchers to study the pharmacokinetics and pharmacodynamics of CP and its metabolites, assess antitumor activity, investigate immunosuppressive effects, and evaluate organ-specific toxicities.

Animal models are used to establish tumor xenografts to assess the antitumor efficacy of this compound alone or in combination with other therapies. For example, murine models have been used to evaluate the efficacy of this compound in combination with CD19 CAR-T cells for treating B-cell hematologic malignancies, demonstrating that a specific pre-conditioning regimen of this compound and fludarabine followed by CAR-T infusion showed the most efficacious therapeutic effect. nih.govresearchgate.net

In vivo studies also investigate the impact of this compound on the immune system. Preclinical murine models have shown that a single low dose of this compound can inhibit regulatory T cells (Tregs) and enhance the immune response to tumor-associated antigens, providing a rationale for its use as a preconditioning agent in cancer vaccine strategies. bmj.com

Animal models are also crucial for studying this compound-induced toxicities, such as osteoporosis. Studies in mice have demonstrated that this compound treatment can induce an osteoporotic phenotype associated with decreased bone formation and resorption. oncotarget.com Pharmacokinetic studies in mice, comparing different blood sampling methods like serial tail vein bleeding and retro-orbital sampling, are conducted to understand the drug's disposition in vivo. benthamopenarchives.com

In combination therapies, in vivo models help determine optimal dosing and schedules. For instance, studies combining this compound with oncolytic viruses in mouse models of glioma have shown that this compound can enhance animal survival and oncolytic virus tumor distribution, suggesting that it may allow for dose reduction of potent oncolytic viruses in clinical trials. nih.govaacrjournals.org

Development of Novel this compound Analogs and Delivery Systems

Efforts to improve the therapeutic profile of this compound include the development of novel analogs with potentially improved efficacy, reduced toxicity, or altered pharmacokinetic properties, as well as the creation of advanced delivery systems to target the drug more effectively to cancer cells and minimize systemic exposure.

The understanding of this compound's mechanism of action, particularly the difference in metabolite formation between in vitro and in vivo conditions, has guided the design of new oxazaphosphorine cytostatics. mdpi.comecronicon.net Novel this compound-like compounds have been developed with the aim of lower toxicity and improved effectiveness, some showing significantly reduced toxicity in preclinical models compared to the parent compound. mdpi.comecronicon.net Examples include aldophosphamide thiazolidines and aldophosphamide perhydrothiazines, which have been synthesized and tested in tumor-bearing mice. ecronicon.net

The synthesis of deuterated analogues of this compound is also employed to probe its microsomal metabolism and study the sites of oxidative metabolism using mass spectrometry. tandfonline.com

In parallel with analog development, research is focused on developing novel delivery systems for this compound. The microencapsulation of this compound into biodegradable polymeric matrices, such as polyhydroxyalkanoate (PHBV) microparticles or chitosan microspheres, is being explored to achieve sustained drug release and potentially reduce adverse effects and administration frequency. researchgate.netiapchem.org Studies have shown that encapsulating this compound can lower its cytotoxicity compared to the pure drug in vitro. iapchem.org

Targeted Delivery Strategies for Metabolites

Targeted delivery of cyclophosphamide or its active metabolites aims to increase their concentration at the disease site while minimizing systemic exposure and associated toxicities. Given that this compound is a prodrug activated in the liver, directly targeting its active metabolites, such as phosphoramide mustard, could offer advantages. However, delivering highly reactive metabolites specifically to tumor cells presents significant challenges.

Research is exploring novel drug delivery systems, such as nanoparticles and liposomes, to encapsulate this compound or its precursors. orientjchem.orgresearchgate.netmdpi.com These systems can be designed to accumulate preferentially in tumor tissue due to the enhanced permeability and retention (EPR) effect or by conjugating them with targeting ligands that bind to receptors overexpressed on cancer cells. mdpi.comnih.govaacrjournals.orgaacrjournals.org For instance, studies have investigated HER2-targeted PEGylated liposomal doxorubicin (MM-302) and found that this compound administration can improve the delivery and antitumor activity of such targeted liposomes in preclinical models. nih.govaacrjournals.orgaacrjournals.org This effect may involve tumor debulking, lowering of interstitial fluid pressure, and increased vascular perfusion, facilitating nanoparticle penetration. nih.govaacrjournals.org

Another approach involves the development of prodrugs that are selectively activated within the tumor microenvironment. This could involve enzymatic activation by enzymes present at higher levels in tumors or activation in response to specific tumor conditions like hypoxia or altered pH. While research in this area is ongoing for various chemotherapeutics, developing tumor-specific activation strategies for this compound's metabolites remains an active area of investigation.

Strategies to Overcome this compound Resistance Mechanisms

Resistance to this compound is a major impediment to its efficacy. Cancer cells can develop resistance through various mechanisms, including increased detoxification of the drug or its metabolites, enhanced DNA repair mechanisms, and alterations in drug targets. nih.govresearchgate.net

One key mechanism of resistance involves increased expression of aldehyde dehydrogenases (ALDH), particularly ALDH1A1 and ALDH3A1. These enzymes can detoxify aldophosphamide, preventing its conversion to the active phosphoramide mustard. dtic.mil Research has identified that ALDH1A1 levels in clinical breast cancers can be of sufficient magnitude to affect the outcome of this compound-based chemotherapy. dtic.mil Strategies to overcome this resistance mechanism include the development of ALDH inhibitors that can be co-administered with this compound to preserve the levels of the active metabolite.

Enhanced DNA repair is another mechanism of resistance. Cancer cells can upregulate pathways that repair the DNA damage induced by phosphoramide mustard, thus mitigating its cytotoxic effects. nih.gov Research is exploring the potential of combining this compound with inhibitors of specific DNA repair pathways to enhance its efficacy in resistant tumors.

Furthermore, the tumor microenvironment can contribute to this compound resistance. Hypoxia and other factors within the microenvironment can influence drug delivery, metabolism, and the sensitivity of cancer cells to the drug. mdpi.com Strategies targeting the tumor microenvironment, such as improving tumor oxygenation or disrupting supportive stromal interactions, are being investigated to enhance this compound's effectiveness.

Integration of Multi-Omics Data in this compound Research

The integration of multi-omics data, including genomics, transcriptomics, proteomics, and metabolomics, is crucial for a comprehensive understanding of this compound's effects and for predicting patient response and resistance. imrpress.comexplorationpub.com By analyzing multiple layers of biological information, researchers can gain insights into the complex molecular mechanisms underlying this compound's action, identify biomarkers of response and resistance, and discover novel therapeutic targets. imrpress.comexplorationpub.com

Genomic studies can identify genetic variations in drug-metabolizing enzymes (e.g., CYP450 enzymes) or drug transporters that influence this compound pharmacokinetics and pharmacodynamics. frontiersin.orgmdpi.comfrontiersin.org Transcriptomic and proteomic analyses can reveal alterations in gene and protein expression profiles associated with response or resistance. imrpress.com Metabolomics can provide insights into how this compound affects cellular metabolism and identify metabolic pathways that contribute to resistance or can be targeted therapeutically. researchgate.netnih.gov

Integrating these different omics datasets through computational approaches can lead to the identification of molecular signatures that predict patient outcomes and guide personalized treatment strategies. imrpress.comexplorationpub.com For example, multi-omics approaches have been used to study the mechanisms by which certain substances might relieve this compound-induced immune injury. researchgate.netnih.gov

Computational Modeling and Simulation of this compound Dynamics

Computational modeling and simulation play a vital role in understanding the complex pharmacokinetics and pharmacodynamics of this compound and its metabolites. frontiersin.orgmdpi.comfrontiersin.orgresearchgate.netnih.gov These models can simulate drug absorption, distribution, metabolism, and excretion (ADME), as well as the interaction of this compound and its metabolites with biological targets and the resulting cellular and systemic effects. researchgate.net

Population pharmacokinetic models, for instance, can describe the variability in this compound and metabolite exposure among different patient populations, such as children, and identify covariates that influence drug clearance. frontiersin.orgmdpi.comfrontiersin.orgnih.gov These models can help optimize dosing strategies to minimize variability in drug exposure and potentially improve efficacy while reducing toxicity. frontiersin.orgmdpi.comfrontiersin.orgresearchgate.net

More complex mechanistic models can incorporate details about enzyme kinetics, transporter activity, and cellular signaling pathways to simulate the intracellular dynamics of this compound metabolites and their effects on DNA damage and cell death. researchgate.net These models can be used to test hypotheses about resistance mechanisms and evaluate the potential impact of combining this compound with other drugs that target specific pathways.

Computational simulations can also aid in the design of targeted delivery systems by predicting the behavior of nanoparticles or liposomes in the body and their accumulation at the tumor site. orientjchem.org This allows for the optimization of nanoparticle properties and targeting strategies.

Immunotherapeutic Combinations and Mechanistic Rationales

This compound has demonstrated immunomodulatory effects, particularly at lower doses, which provides a rationale for combining it with immunotherapy. nih.govaacrjournals.orgnih.govmdpi.comeur.nlaacrjournals.orgtandfonline.com this compound can influence the immune system by depleting regulatory T cells (Tregs), which suppress antitumor immune responses, and by promoting the proliferation and activation of effector T cells. nih.govaacrjournals.orgnih.govmdpi.comeur.nlaacrjournals.orgtandfonline.com

Combining this compound with immune checkpoint inhibitors (ICIs), such as antibodies targeting PD-1 or CTLA-4, is a promising strategy being investigated in preclinical and clinical settings. nih.govmdpi.comaacrjournals.orgcancer.govresearchgate.net The rationale is that this compound can create a more favorable tumor microenvironment for the action of ICIs by reducing immunosuppressive cells and enhancing antitumor immunity. nih.govmdpi.comaacrjournals.orgresearchgate.net Studies have shown that this compound can enhance the efficacy of ICIs in various tumor models, including melanoma and non-small cell lung cancer. aacrjournals.orgcancer.govresearchgate.net

Q. What validated analytical methods are recommended for quantifying cyclophosphamide in pharmaceutical formulations?

Reverse-phase HPLC with UV detection is widely used, employing a C18 column and mobile phase (e.g., methanol-phosphate buffer). Ethylparaben serves as an internal standard to normalize peak responses. Validation parameters include linearity (1–100 µg/mL), precision (RSD <2%), and recovery (98–102%) .

Q. How does this compound exert its alkylating mechanism, and what experimental models confirm this?

this compound is metabolized by hepatic CYP450 enzymes into phosphoramide mustard, which cross-links DNA. In vitro studies using human liver microsomes or cell lines (e.g., Jurkat T-cells) validate metabolite formation via LC-MS. In vivo, murine tumor models demonstrate dose-dependent apoptosis via TUNEL assays .

Q. What are standard protocols for assessing this compound’s cytotoxicity in preclinical studies?

Use clonogenic assays (colony-forming efficiency) or MTT assays at 24–72 hours post-treatment. IC₅₀ values vary by cell type (e.g., 511 µM for AChE inhibition in neuroblastoma models). Include positive controls (e.g., cisplatin) and normalize to vehicle-treated cells .

Q. How should researchers account for this compound’s pharmacokinetic variability in animal models?

Adjust doses based on species-specific metabolic rates (e.g., 100–200 mg/kg in mice vs. 10–20 mg/kg in rats). Monitor plasma levels via LC-MS/MS and consider nephroprotective agents (e.g., mesna) to reduce hemorrhagic cystitis risk .

Advanced Research Questions

Q. What experimental designs resolve contradictions in this compound’s immunomodulatory effects at low vs. high doses?

Low-dose regimens (e.g., 50 mg/kg in mice) deplete regulatory T cells (Tregs) via FoxP3/GITR downregulation, enhancing antitumor immunity. High doses (>150 mg/kg) induce broad immunosuppression. Use flow cytometry (CD4+/CD25+/FoxP3+ markers) and adoptive transfer models to isolate Treg functionality .

Q. How can combination therapies with this compound be optimized to minimize resistance in solid tumors?

Sequential dosing with immune checkpoint inhibitors (e.g., anti-PD-1) amplifies immunogenic cell death. In HER2+ breast cancer, docetaxel/cyclophosphamide (TC) improved 7-year OS (87% vs. 82% for AC regimen) via synergistic microtubule disruption and DNA damage .

Q. What methodologies address conflicting data on this compound’s role in autoimmune disease models?

Dose-titration studies in collagen-induced arthritis (CIA) models show bimodal effects: low doses suppress inflammation (reduced IL-17/IFN-γ), while high doses exacerbate it. Use cytokine multiplex assays and single-cell RNA-seq to map Th17/Treg balance .

Q. How should long-term survival analysis be conducted in this compound-based clinical trials?

Apply Kaplan-Meier estimators with log-rank tests for OS/DFS comparisons. In the US Oncology 9735 trial, TC’s hazard ratio for OS was 0.69 (95% CI: 0.50–0.97) with median 7-year follow-up. Stratify by age, hormone receptor status, and HER2 expression .

Q. What are the mechanistic implications of this compound-induced oxidative stress in pediatric populations?

Pediatric dosing (e.g., 1.5–2 g/m² in ALL protocols) requires glutathione depletion monitoring via GSH/GSSG assays. Oxidative DNA damage (8-OHdG levels) correlates with secondary malignancy risk, necessitating long-term follow-up .

Q. How do tumor microenvironment (TME) alterations influence this compound efficacy in adoptive cell therapy?

Preconditioning with this compound (e.g., 60 mg/kg) enhances tumor-infiltrating lymphocyte (TIL) engraftment by depleting myeloid-derived suppressor cells (MDSCs). Use multiplex IHC (CD33+/CD11b+ markers) and cytokine profiling (IL-2, IFN-γ) to validate TME remodeling .