Anticancer Prodrug Cyclophosphamide Research Articles

Anti-Cancer Prodrug Cyclophosphamide Exerts Thrombogenic Effects on Human Venous Endothelial Cells Independent of CYP450 Activation-Relevance to Thrombosis.

Cancer patients are at a very high risk of serious thrombotic events, often fatal. The causes discussed include the detachment of thrombogenic particles from tumor cells or the adverse effects of chemotherapeutic agents. Cytostatic agents can either act directly on their targets or, in the case of a prodrug approach, require metabolization for their action. Cyclophosphamide (CPA) is a widely used cytostatic drug that requires prodrug activation by cytochrome P450 enzymes (CYP) in the liver. We hypothesize that CPA could induce thrombosis in one of the following ways: (1) damage to endothelial cells (EC) after intra-endothelial metabolization; or (2) direct damage to EC without prior metabolization. In order to investigate this hypothesis, endothelial cells (HUVEC) were treated with CPA in clinically relevant concentrations for up to 8 days. HUVECs were chosen as a model representing the first place of action after intravenous CPA administration. No expression of CYP2B6, CYP3A4, CYP2C9 and CYP2C19 was found in HUVEC, but a weak expression of CYP2C18 was observed. CPA treatment of HUVEC induced DNA damage and a reduced formation of an EC monolayer and caused an increased release of prostacyclin (PGI2) and thromboxane (TXA) associated with a shift of the PGI2/TXA balance to a prothrombotic state. In an in vivo scenario, such processes would promote the risk of thrombus formation.

Pharmacokinetics and Pharmacogenetics of Cyclophosphamide in a Neonate and Infant Childhood Cancer Patient Population.

Infants and young children represent an important but much understudied childhood cancer patient population. The pharmacokinetics and pharmacogenetics of the widely used anticancer prodrug cyclophosphamide were investigated in children <2 years of age. Concentrations of cyclophosphamide and selected metabolites were determined in patients administered cyclophosphamide at doses ranging from 100–1500 mg/m2 (5–75 mg/kg), with various infusion times as determined by the standard treatment regimen that each patient was receiving. Polymorphisms in genes including CYP2B6 and CYP2C19 were investigated. Data generated for cyclophosphamide were analysed using a previously published population pharmacokinetic model. Cyclophosphamide pharmacokinetics was assessed in 111 samples obtained from 25 patients ranging from 4–23 months of age. The average cyclophosphamide clearance for the patients was 46.6 mL/min/m2 (ranging from 9.4–153 mL/min/m2), with marked inter-patient variability observed (CV 41%). No significant differences in cyclophosphamide clearance or exposure (AUC) were observed between patient groups as separated by age or body weight. However, marked differences in drug clearance and metabolism were noted between the current data in children <2 years of age and recently published results from a comparable study conducted by our group in older children, which reported significantly lower cyclophosphamide clearance values and metabolite exposures using the same population pharmacokinetic model for analysis. Whilst this study demonstrates no significant differences in cyclophosphamide clearance in patients <2 years, it highlights large differences in dosing protocols across tumour types. Furthermore, the study suggests marked differences in cyclophosphamide clearance in children less than two years of age as compared to older patients.

Predicting Metabolism-Related Drug-Drug Interactions Using a Microphysiological Multitissue System.

Drug-drug interactions (DDIs) occur when the pharmacological activity of one drug is altered by a second drug. As multimorbidity and polypharmacotherapy are becoming more common due to the increasing age of the population, the risk of DDIs is massively increasing. Therefore, in vitro testing methods are needed to capture such multiorgan events. Here, a scalable, gravity-driven microfluidic system featuring 3D microtissues (MTs) that represent different organs for the prediction of drug-drug interactions is used. Human liver microtissues (hLiMTs) are combined with tumor microtissues (TuMTs) and treated with drug combinations that are known to cause DDIs in vivo. The testing system is able to capture and quantify DDIs upon co-administration of the anticancer prodrugs cyclophosphamide or ifosfamide with the antiretroviral drug ritonavir. Dosage of ritonavir inhibits hepatic metabolization of the two prodrugs to different extents and decreases their efficacy in acting on TuMTs. The flexible MT compartment design of the system, the use of polystyrene as chip material, and the assembly of several chips in stackable plates offer the potential to significantly advance preclinical substance testing. The possibility of testing a broad variety of drug combinations to identify possible DDIs will improve the drug development process and increase patient safety.

Encapsulation of liver microsomes into a thermosensitive hydrogel for characterization of drug metabolism and toxicity

This study reported the encapsulation of liver microsomes into a thermosensitive hydrogel to characterize drug metabolism and predict drug effects. Pluronic®F-127 (F127) and acrylamide-bisacrylamide (Acr-Bis) were utilized as the two precursors. After chemical crosslinking catalyzed by ammonium persulfate (APS) and N,N,N′,N′-tetramethylethylenediamine (TEMED), the resulting Pluronic F127-acrylamide-bisacrylamide (FAB) hydrogel could encapsulate microsomes at 4 °C and facilitate metabolic reactions at 37 °C. The gel morphology at different Acr-Bis concentrations was characterized using field emission scanning electron microscopy (FE-SEM). Higher concentrations of Acr-Bis could lead to higher degrees of cross-linking of the gel. A fluorescent staining assay was subsequently used to demonstrate successful encapsulation of microsomes into the gel as well as the free diffusion process of micromolecular substrates. The thermosensitivity of the FAB gel was studied using swelling ratio and protein release assay to verify its ability to encapsulate microsomes. The metabolic activity of microsomes encapsulated in gels was investigated by detecting the metabolites of FDA-approved substrates, including dextromethorphan, chlorzoxazone and testosterone. Compared with the traditional method of microsomal incubation, the FAB gel maintained 60%–70% of microsome activity. Lastly, the classic anticancer prodrug cyclophosphamide (CTX) was chosen as a model drug for the study of drug metabolism and the prediction of drug effects. When the microsomes encapsulated in the FAB gel were used in the cell culture system, CTX induced a higher level of apoptosis in MCF-7 cells compared with traditional microsomes.

Challenges and Opportunities of Cytochrome P450-Mediated Phytoremediation

Although environmental pollution was first realized to be a threat to public health in the latter part of the 20th century, events in the 21st century have served to increase the public awareness of the impacts of environmental pollution. Several communities, including the scientific community, initiated dialogues to help protect the planet from environmental pollution. Further, scientific community initiated basic and applied research in an effort to decontaminate soil, water, and air pollutions arising from industrial and petroleum wastes, as well as from the use of pesticides and herbicides. They utilized plant enzymes, as well as bacterial, fungal, and mammalian enzymes cloned in plants (transgenic plants), to detoxify soil and water contaminations (phytoremediation). Although there was a slow increase in research publications of phytoremediation from 1990-2000, a significant increase in their publications from 2001-2008 was observed (Figure 1). Interestingly, a decline in research publications since 2009 was observed, which suggests that there is a challenge in this type of research for further improvements in phytoremediation. A similar trend was also observed with cytochrome P450 (CYP)-mediated phytoremediation (Figure 1). In this editorial, we will discuss the challenges and opportunities of CYP-mediated phytoremediation. The discussion in this editorial is based on a recent review [1]. Figure 1 Number of publications from 1990 to 2012 using the key words PubMed (control), phytoremediation, and phytoremediation+cytochrome P450 (CYP). Left Y-axis represents number of publications for PubMed and phytoremediation and right Y-axis represents number ... CYP family of enzymes involved in the metabolism and detoxification of numerous xenobiotics, including herbicides, pesticides, and industrial pollutants, such as pharmaceuticals, petrochemicals, polycyclic aromatic hydrocarbons (PAHs), and polychlorinated benzene (PCBs) [2]. The catalytic versatility of CYP enzymes have led to significant interest in utilizing them as biocatalysts for phytoremediation. In the last decade there has been an increase in the utilization of CYP biocatalysts for creating herbicide-resistant plants and for the detoxification of soil and water contaminants. The use of transgenic plants expressing bacterial and mammalian CYP enzymes for phytoremediation is crucial because plant CYP enzymes are poor in detoxifying these contaminants [3], whereas bacterial and mammalian CYP enzymes have the capability to detoxify herbicides and industrial pollutants [4,5]. However, the major limitation of mammalian CYP enzymes is that they have relatively low turn-over, enzyme stability, and expression in plants. In contrast, many bacterial CYP enzymes display high levels of activity and stability; however, they metabolize limited number of pollutants. These are the major challenges in the utilization of CYP enzymes for phytoremediation that may have led to a decrease in research publications for CYP-mediated phytoremediation since 2009 (Figure 1). However, these challenges also bring an opportunity to expand innovative research towards engineering of CYP enzymes for the improvement of their substrate diversity, activity, stability, and expression in plants. Since the beginning of 21st century, significant progress has been made, especially by Arnold and colleagues, in designing bacterial CYP enzymes [6,7]. For example, a bacterial CYPBM3 enzyme has been engineered for novel activity towards hydroxylation of many non-natural alkanes, such as asoctane, hexane, propane, butane, and ethane, as well as other non-alkyl substrates, such as benzene, propylbenzene, and naphthalene. Similarly, another bacterial CYPCAM enzyme has been engineered for efficient oxidation of various alkanes (butane, pentane, hexane, and heptanes), halogenated hexanes, PCBs, and PAHs. These engineered CYPBM3 and CYPCAM enzymes can potentially be used to create transgenic plants for possible phytoremediation of these toxic compounds. Further, several mammalian CYP enzymes have been engineered to improve activity, stability, and heterologous expression [8]. For examples: 1) Guengerich and colleagues have successfully engineered human CYP2A6 enzyme for increased activity with indigo compounds and cloned it into tobacco plants that produced the precursor of indole dyes; 2) Kumar and Halpert groups have engineered Rat CYP2B1 and canine CYP2B11 enzymes for enhanced activity with several compounds, including anti-cancer prodrugs cyclophosphamide and ifosfamide; 3) Gillam and colleagues have designed CYP2C enzymes for novel activity with 6′-deoxyluciferin and diclofenac. Although these mammalian CYP enzymes were not engineered for enhanced activity with herbicides, industrial contaminants, or petroleum compounds, they provide a proof-of-concept that mammalian CYP enzymes can be engineered for phytoremediation of these compounds. In conclusion, phytoremediation of soil and water contaminants arising from herbicides, pesticides, and industrial pollutants has been identified to be critically important to clean the environment and improve air and water qualities. Although generation of CYP-based transgenic plants has the potential to serve this purpose, certain limitations of CYP enzymes posed challenges in their application in phytoremediation. Thus, there is a critical need to expand the basic and applied research in designing CYP biocatalysts for phytoremediation and therefore additional effort is required from CYP enzyme and protein engineering communities towards this.

Re-engineering cytochrome P450 2B11dH for enhanced metabolism of several substrates including the anti-cancer prodrugs cyclophosphamide and ifosfamide

Based on recent directed evolution of P450 2B1, six P450 2B11 mutants at three positions were created in an N-terminal modified construct termed P450 2B11dH and characterized for enzyme catalysis using five substrates. Mutant I209A demonstrated a 3.2-fold enhanced k cat/ K m for 7-ethoxy-4-trifluoromethylcourmarin O-deethylation, largely due to a dramatic decrease in K m (0.72 μM vs. 18 μM). I209A also demonstrated enhanced selectivity for testosterone 16β-hydroxylation over 16α-hydroxylation. In contrast, V183L showed a 4-fold increased k cat for 7-benzyloxyresorufin debenzylation and a 4.7-fold increased k cat/ K m for testosterone 16α-hydroxylation. V183L also displayed a 1.7-fold higher k cat/ K m than P450 2B11dH with the anti-cancer prodrugs cyclophosphamide and ifosfamide, resulting from a ∼4-fold decrease in K m. Introduction of the V183L mutation into full-length P450 2B11 did not enhance the k cat/ K m. Overall, the re-engineered P450 2B11dH enzymes exhibited enhanced catalytic efficiency with several substrates including the anti-cancer prodrugs.

Human P450 Microarrays for In Vitro Toxicity Analysis: Toward Complete Automation of Human Toxicology Screening

The development of a tool that can provide early-stage predictive toxicology data may accelerate the identification of safer drug candidates, and thereby improve the clinical progression of drug candidates to pharmaceuticals. Such a system would require an accurate and reliable technique that is amenable to the large number of drug candidates that must be screened in the lead discovery and optimization stages of drug development. A key component of predictive toxicology is the ability to harness the metabolite-generating capacity of human cytochromes P450, which are involved in first-pass drug metabolism function of the liver. We have miniaturized P450 catalysis into a microarray format consisting of up to 11,200 isolated P450 reactions, each in 5 nL sol-gel spots, on a single functionalized glass microscope-size biochip. This dramatic scale down from more conventional 96 and 384-well plate scales (at least a 1000-fold reduction in volume) did not adversely affect P450 catalytic activity. Based on the functionality of the P450-containing microarray, we developed the metabolizing enzyme toxicology assay Chip (MetaChip), which combines high-throughput P450 catalysis with cell-based screening on a microscale platform. Proof of concept was demonstrated using anticancer prodrugs cyclophosphamide and Tegafur, as well as the analgesic acetaminophen. The MetaChip may provide a high-throughput microscale alternative to currently used in vitro methods for human metabolism and toxicology screening.

Effects of hypoxia and limited diffusion in tumor cell microenvironment on bystander effect of P450 prodrug therapy

Cytochrome P450 (CYP) enzyme 2B1 metabolizes the anticancer prodrug cyclophosphamide (CPA) to 4-hydroxy-CPA, which decomposes to the cytotoxic metabolites acrolein and phosphoramide mustard. We have evaluated the bystander cytotoxicity of CPA in combination with CYP2B1 gene-directed enzyme prodrug therapy using a cell culture-based agarose overlay technique. This method mimics the tumor microenvironment by limiting the diffusion of metabolites and by reducing the oxygen concentration to levels similar to those found in solid tumors. Under these conditions, the CYP activity of CYP2B1-expressing tumor cells was decreased by 80% compared to standard aerobic conditions. Despite this decrease in metabolic activity, a potent bystander effect was observed, resulting in up to 90% killing by CPA of a tumor cell population comprised of only approximately 20% CYP-expressing tumor cells. Similarly, transient transfection of a small fraction ( approximately 14%) of a human hepatoma Huh7 cell population with a CYP2B1 expression plasmid followed by short-term treatment with CPA (5 h) led to an eradication of 95% of the cells. No such bystander effect was observed without the agarose overlay. These findings suggest that the agarose overlay technique is very useful as an in vitro test system for investigation of the bystander effect of CYP/CPA and other enzyme/prodrug combinations under conditions that mimic the hypoxic conditions present in solid tumors in vivo.

Directed evolution of mammalian cytochromes P450 for investigating the molecular basis of enzyme function and generating novel biocatalysts

Xenobiotic-metabolizing mammalian cytochromes P450 with their catalytic versatility and broad substrate specificity offer the possibility of widespread applications in industrial synthesis, medicine, and bioremediation. However, the requirement for NADPH-cytochrome P450 reductase, often cytochrome b5, and an expensive cofactor, NADPH, complicates the design of mammalian P450 enzymes as biocatalysts. We have developed a directed evolution approach for a number of mammalian P450s using fluorescence-based marker substrates in a peroxide-supported in vitro screening system. Directed evolution of rat 2B1 using 7-ethoxy(trifluoromethyl)coumarin yielded several mutants with enhanced catalytic efficiency toward multiple substrates including the anti-cancer prodrugs cyclophosphamide and ifosfamide (Kumar et al., J. Biol. Chem. 280, 19569–19575, 2005). Furthermore, the evolved 2B1 mutant showed a significant increase in affinity for H2O2 and enzyme stability. A similar approach using 7-benzyloxyquinoline yielded several human P450 3A4 mutants with altered properties including increased affinity for H2O2. More recently, this approach was also developed with human P450s from the 2C subfamily. In conclusion, the successful application of directed evolution with several mammalian P450s suggests that this approach can be expanded to more targeted substrates of environmental, industrial, and medical importance to design more efficient P450 biocatalysts. [NIEHS pilot grant ES06676 (S.K.), NIH Grants ES03619 and GM54995, and Center grant ES06676 (J.R.H.)]

Population Pharmacokinetics of Cyclophosphamide and Its Metabolites 4-Hydroxycyclophosphamide, 2-Dechloroethylcyclophosphamide, and Phosphoramide Mustard in a High-Dose Combination With Thiotepa and Carboplatin

The anticancer prodrug cyclophosphamide (CP) is activated by the formation of 4-hydroxycyclophosphamide (4OHCP), which decomposes into phosphoramide mustard (PM). This activation pathway is inhibited by thiotepa. CP is inactivated by formation of 2-dechloroethylcyclophosphamide (2DCECP). The aim of this study was to develop a population pharmacokinetic model describing the complex pharmacokinetics of CP, 4OHCP, 2DCECP, and PM when CP is administered in a high-dose combination with thiotepa and carboplatin. Patients received a combination of CP (1000-1500 mg/m/d), carboplatin (265-400 mg/m/d), and thiotepa (80-120 mg/m/d) administered in short infusions over 4 days. Twenty blood samples were collected per patient per course. Concentrations of CP, 4OHCP, 2DCECP, PM, thiotepa, and tepa were determined in plasma. Using NONMEM, an integrated population pharmacokinetic model was used to describe the pharmacokinetics of CP, 4OHCP, 2DCECP, and PM, including the already described processes of autoinduction of CP and the interaction with thiotepa. Data were available on 35 patients (70 courses). The pharmacokinetics of CP were described with a 2-compartment model, and those of 4OHCP, 2DCECP, and PM with 1-compartment models. Before onset of autoinduction, it was assumed that CP is eliminated through a noninducible pathway accounting for 20% of total CP clearance, whereas 2 inducible pathways resulted in formation of 4OHCP (75%) and 2DCECP (5%). It was assumed that 4OHCP was fully converted to PM. Induction of CP metabolism was mediated by 2 hypothetical amounts of enzyme whose quantities increased in time in the presence of CP (kenz=0.0223 and 0.0198 hours). Induction resulted in an increased formation of 4OHCP (approximately 50%), PM (approximately 50%), and 2DCECP (approximately 35%) during the 4-day course, and concomitant decreased exposure to CP (approximately 50%). The formation of 2DCECP was not inhibited by thiotepa. Apparent volumes of distribution of CP, PM, and 2DCECP could be estimated being 43.7, 55.5, and 18.5 L, respectively. Exposure to metabolites varied up to 9-fold. The complex population pharmacokinetics of CP, 4OHCP, 2DCECP, and PM in combination with thiotepa and carboplatin has been established and may form the basis for further treatment optimization with this combination.

Directed Evolution of Mammalian Cytochrome P450 2B1: MUTATIONS OUTSIDE OF THE ACTIVE SITE ENHANCE THE METABOLISM OF SEVERAL SUBSTRATES, INCLUDING THE ANTICANCER PRODRUGS CYCLOPHOSPHAMIDE AND IFOSFAMIDE

Cytochrome P450 2B1 has been subjected to directed evolution to investigate the role of amino acid residues outside of the active site and to engineer novel, more active P450 catalysts. A high throughput screening system was developed to measure H(2)O(2)-supported oxidation of the marker fluorogenic substrate 7-ethoxy-4-trifluoromethylcoumarin (7-EFC). Random mutagenesis by error-prone polymerase chain reaction and activity screening were optimized using the L209A mutant of P450 2B1 in an N-terminally modified construct with a C-terminal His tag (P450 2B1dH). Two rounds of mutagenesis and screening and one subcloning step yielded the P450 2B1 quadruple mutant V183L/F202L/L209A/S334P, which demonstrated a 6-fold higher k(cat) than L209A. Further random or site-directed mutagenesis did not improve the activity. When assayed in an NADPH-supported reconstituted system, V183L/L209A demonstrated lower 7-EFC oxidation than L209A. Therefore, F202L/L209A/S334P was generated, which showed a 2.5-fold higher k(cat)/K(m) for NADPH-dependent 7-EFC oxidation than L209A. F202L/L209A/S334P also showed enhanced catalytic efficiency with 7-benzyloxyresorufin, benzphetamine, and testosterone, and a 10-fold increase in stereoselectivity for testosterone 16alpha-versus 16beta-hydroxylation compared with 2B1dH. Enhanced catalytic efficiency of F202L/L209A/S334P was also retained in the full-length P450 2B1 background with 7-EFC and testosterone as substrates. Finally, the individual mutants were tested for metabolism of the anti-cancer prodrugs cyclophosphamide and ifosfamide. Several of the mutants showed increased metabolism via the therapeutically beneficial 4-hydroxylation pathway, with L209A/S334P showing 2.8-fold enhancement of k(cat)/K(m) with cyclophosphamide and V183L/L209A showing 3.5-fold enhancement with ifosfamide. Directed evolution can thus be used to enhance P450 2B1 catalytic efficiency across a panel of substrates and to identify functionally important residues distant from the active site.

Activation of the anticancer prodrugs cyclophosphamide and ifosfamide: identification of cytochrome P450 2B enzymes and site-specific mutants with improved enzyme kinetics.

Cyclophosphamide (CPA) and ifosfamide (IFA) are oxazaphosphorine anticancer prodrugs metabolized by two alternative cytochrome P450 (P450) pathways, drug activation by 4-hydroxylation and drug inactivation by N-dechloroethylation, which generates the neurotoxic and nephrotoxic byproduct chloroacetaldehyde. CPA and IFA metabolism catalyzed by P450s 2B1, 2B4, 2B5, and seven site-specific 2B1 mutants was studied in a reconstituted Escherichia coli expression system to identify residues that contribute to the unique activities and substrate specificities of these enzymes. The catalytic efficiency of CPA 4-hydroxylation by rat P450 2B1 was 10- to 35-fold higher than that of rabbit P450 2B4 or 2B5. With IFA, approximately 50% of metabolism proceeded via N-dechloroethylation for 2B1 and 2B4, whereas CPA N-dechloroethylation corresponded to only approximately 3% of total metabolism (2B1) or was absent (2B4, 2B5). Improved catalytic efficiency of CPA and IFA 4-hydroxylation was obtained upon substitution of 2B1 Ile-114 by Val, and replacement of Val-363 by Leu or Ile selectively suppressed CPA N-dechloroethylation >or=90%. P450 2B1-V367A, containing the Ala replacement found in 2B5, exhibited only approximately 10% of wild-type 2B1 activity for both substrates. Canine P450 2B11, which has Val-114, Leu-363, and Val-367, was therefore predicted to be a regioselective CPA 4-hydroxylase with high catalytic efficiency. Indeed, P450 2B11 was 7- to 8-fold more active as a CPA and IFA 4-hydroxylase than 2B1, exhibited a highly desirable low K(m) (80-160 microM), and catalyzed no CPA N-dechloroethylation. These findings provide insight into the role of specific P450 2B residues in oxazaphosphorine metabolism and pave the way for gene therapeutic applications using P450 enzymes with improved catalytic activity toward these anticancer prodrug substrates.

Regioselectivity of CYP2B6: homology modeling, molecular dynamics simulation, docking.

Human cytochrome P450 (CYP) 2B6 activates the anticancer prodrug cyclophosphamide (CPA) by 4-hydroxylation. In contrast, the same enzyme catalyzes N-deethylation of a structural isomer, the prodrug ifosfamide (IFA), thus causing severe adverse drug effects. To model the molecular interactions leading to a switch in regioselectivity, the structure of CYP2B6 was modeled based on the structure of rabbit CYP2C5. We modeled the missing 22-residue loop in CYP2C5 between helices F and G (the F-G loop), which is not resolved in the X-ray structure, by molecular dynamics (MD) simulations using a simulated annealing protocol. The modeled conformation of the loop was validated by unconstrained MD simulations of the complete enzymes (CYP2C5 and CYP2B6) in water for 70 and 120 ps, respectively. The simulations were stable and led to a backbone r.m.s. deviation of 1.7 A between the two CYPs. The shape of the substrate binding site of CYP2B6 was further analyzed. It consists of three well-defined hydrophobic binding pockets adjacent to the catalytic heme. Size, shape and hydrophobicity of these pockets were compared to the shapes of the two structurally isomeric substrates. In their preferred orientation in the binding site, both substrates fill all three binding pockets without repulsive interactions. The distance to the heme iron is short enough for 4-hydroxylation and N-deethylation to occur for CPA and IFA, respectively. However, if the substrates are docked in the non-preferred orientation (such that 4-hydroxylation and N-deethylation would occur for IFA and CPA, respectively), one pocket is left empty, and clashes were observed between the substrates and the enzyme.

Impact of liver P450 reductase suppression on cyclophosphamide activation, pharmacokinetics and antitumoral activity in a cytochrome P450-based cancer gene therapy model.

The effect of the antithyroid drug methimazole (MMI) on cytochrome P450/P450 reductase-dependent activation of the anti-cancer prodrug cyclophosphamide (CPA) was investigated in a rat model of P450 prodrug activation-based cancer gene therapy. MMI treatment decreased the expression of hepatic P450 reductase by approximately 75% but did not alter P450 reductase levels in a 9L gliosarcoma growing in vivo as a subcutaneous solid tumor. In a pharmacokinetic study, MMI treatment significantly decreased the peak plasma concentration of the active, P450-generated metabolite 4-hydroxy-CPA, from 84.1 to 57.8 microM, and substantially prolonged its apparent half-life, from 25.4 to 54.3 minutes. The area under the plasma concentration x time curve and clearance values for 4-hydroxy-CPA were largely unchanged, however, indicating that MMI decreases the rate but not the overall extent of hepatic CPA activation. MMI alleviated some of the systemic toxicities of CPA treatment, as judged by the moderation of CPA-induced body weight loss and hematuria. The impact of MMI on CPA antitumoral activity was evaluated in rats implanted with 9L tumors transduced with P450 reductase in combination with the CPA-activating P450 2B1, which confers the capacity for intratumoral prodrug activation and leads to markedly enhanced chemosensitivity. CPA given as a single, subtherapeutic dose of 75 mg/kg resulted in a 13.8 day growth delay, whereas CPA in combination with MMI increased the growth delay to 17.4 days. By contrast, a tumor growth delay of only 3.4 days was observed in animals bearing 9L wild-type tumors given the same drug combination. We conclude that the selective reduction of liver P450 reductase after MMI treatment decreases the rate of hepatic drug activation and the host toxicity of CPA without loss of the antitumoral effect, thus increasing the therapeutic index of CPA in a P450-based cancer gene therapy model, where CPA undergoes localized drug activation at its intratumoral site of action.

Expression of cytochrome P 450 3A enzymes in human lung: a combined RT-PCR and immunohistochemical analysis of normal tissue and lung tumours.

We have previously demonstrated expression of cytochrome P 450 3A (CYP3A) protein in pulmonary carcinomas and surrounding normal tissue, using immunohistochemistry. These results suggested that different CYP3A enzymes may be expressed in normal and tumour tissue. Therefore, the aim of the present study was to identify specific CYP3A enzymes expressed in normal human lung and lung tumours. Both normal lung tissue and tumour tissue from eight patients was analyzed for CYP3A4, CYP3A5 and CYP3A7 mRNA using a specific RT-PCR (reverse transcriptase-polymerase chain reaction) method. Identical samples were subjected to immunohistochemical analysis of CYP3A protein. CYP3A5 was the major enzyme of the CYP3A subfamily present at the mRNA level in both normal human lung and lung tumours. CYP3A5 mRNA was detected in normal lung tissue in all eight cases and in tumour tissue in four cases. CYP3A7 mRNA was detected in five cases in normal tissue and in one tumour. Notably, no CYP3A4 mRNA was found in any of the samples. Immunohistochemical staining for CYP3A protein was found in normal lung tissue in each case. Interestingly, all pulmonary carcinomas showed immunostaining for CYP3A, while mRNA for CYP3A enzymes was found in only four cases. In summary, our study indicates a specific expression pattern of the members of the CYP3A subfamily in normal human lung and lung tumours. These findings have potential clinical significance, since it has been recently shown that CYP3A5 catalyzes the activation of the anticancer pro-drugs cyclophosphamide and ifosfamide. Thus, local activation of these agents may take place in pulmonary carcinomas and surrounding normal tissues.

NMR spectroscopic studies of intermediary metabolites of cyclophosphamide. A comprehensive kinetic analysis of the interconversion of cis- and trans-4-hydroxycyclophosphamide with aldophosphamide and the concomitant partitioning of aldophosphamide between irreversible fragmentation and reversible conjugation pathways.

Multinuclear (31P, 13C, 2H, and 1H) Fourier-transform NMR spectroscopy, with and without isotopically enriched materials, was used to identify and quantify, as a function of time, the following intermediary (short-lived) metabolites of the anticancer prodrug cyclophosphamide (1, Scheme I): cis-4-hydroxycyclophosphamide (cis-2), its trans isomer (trans-2), aldophosphamide (3), and its aldehyde-hydrate (5). Under a standard set of reaction conditions (1 M 2,6-dimethylpyridine buffer, pH 7.4, 37 degrees C), the stereospecific deoxygenation of synthetic cis-4-hydroperoxycyclophosphamide (cis-12, 20 mM) with 4 equiv of sodium thiosulfate (Na2S2O3) afforded, after approximately 20 min, a "pseudoequilibrium" distribution of cis-2, 3, 5, and trans-2, i.e., the relative proportions of these reactants (57:4:9:30, respectively) remained constant during their continual disappearance. NMR absorption signals indicative of "iminophosphamide" (8) and enol 6 were not detected (less than 0.5-1% of the synthetic metabolite mixture). A computerized least-squares fitting procedure was applied to the individual 31P NMR derived time courses for conversion of cis-2, 3 plus 5 (i.e., "3"), and trans-2 into acrolein and phosphoramide mustard (4), the latter of which gave an expected array of thiosulfate S-alkylation products (e.g., 16) and other phosphorus-containing materials derived from secondary decomposition reactions. This kinetic analysis gave the individual forward and reverse rate constants for the apparent tautomerization processes, viz., cis-2 in equilibrium "3" in equilibrium trans-2, as well as the rate constant (k3) for the irreversible fragmentation of 3. The values of k3 at pH 6.3, 7.4, and 7.8 were equal to 0.030 +/- 0.004, 0.090 +/- 0.008, and 0.169 +/- 0.006 min-1, respectively. Replacement of the HC(O)CH2 moiety n 3 with HC(O)CD2 led to a primary kinetic isotope effect (kH/kD = 5.6 +/- 0.4) for k3. The apparent half-lives (tau 1/2) for cis-2, "3", and trans-2 under the standard reaction conditions, at "pseudoequilibrium" (constant ratio of cis-2/"3"/trans-2), were each equal to approximately 38 min, which is considerably shorter than the widely cited colorimetrically derived half-lives reported by earlier investigators. The values of tau 1/2 for cis-2, "3", and trans-2 were affected by pH in the same manner as that found for k3 but were relatively insensitive to the presence of either K+, Na+, Ca2+, or Mg2+. The presence of certain primary amines led to marked decreases in tau 1/2 and, in some cases, the formation of acyclic adducts of aldehyde 3.(ABSTRACT TRUNCATED AT 400 WORDS)