PROTECTIVE ROLE OF AQUEOUS LEAF EXTRACT OF Vernonia amygdalina Del. (Asteraceae) AND Ocimum gratissimum Linn (Lamiaceae) IN CYCLOPHOSPHAMIDE INDUCED UROTOXICITY AND MYELOSUPPRESSION
Cyclophosphamide (CP) is one of the most potent and widely used alkylating anticancer agents. Urotoxicity and myelosuppression is known as the most prevailing dose-limiting toxicity associated with CP. In the present study, the protective potential of Vernonia amygdalina and Ocimum gratissimum aqueous leaf extracts in CP-induced urotoxicity and myelosupression were evaluated using biochemical and histopathological approaches. Sodium -2-macarptoethane sulfonate (MESNA) was used as a positive control.
Forty (40) male Sprague-Dawley outbred albino rats weighing between 130 g – 200 g were randomly separated into eight different groups (n=5). Rats in group 1 received only normal saline orally for gavage for ten consecutive days. Animals in group two were injected with
CP only on the tenth day intraperitoneally (i.p) at 200 mg/kg body weight. Animals in group 3 were given MESNA (67 mg/kg) and CP (200 mg/kg) i.p on the tenth day at 5 minutes interval. Rats in groups 4 and 5 received two different doses of O. gratissimum orally by gavage at 250 mg/kg and 500 mg/kg respectively for ten consecutive days before administering CP (200 mg/kg) on the tenth day. Rats in group 6 and 7 received different doses of V. amygdalina orally by gavage at 250 mg/kg and 500 mg/kg respectively for ten consecutive days before administering CP (200 mg/kg) on the tenth day. Rats in group (8) received combination of V. amygdalina and O. gratissimum at a dose of 250 mg/kg each for ten consecutive before administering CP (200 mg/kg) on the tenth day. Results showed that the extract of V. amygdalina protected significantly (P < 0.05) the urothelium and the myeloid system as observed in the biochemical and hematological parameters evaluated. This protection is comparable to MESNA, but MESNA protection was not adequate to prevent myelosupression as observed with V. amygdalina. O. gratissimum did not show significant protection of the urothelium and myeloid system. The protective effects of V. amygdalina was further evident through decreased histopathological alteration of the urinary bladder, kidney and liver tissues unlike the CP and O. gratissimum treated groups. The result of the present study revealed that aqueous leaf extract of V. amygdalina has the potential to prevent urotoxicity and myelosuppression induced by CP and thus can be used as therapeutic adjuvant in the management of CP and other oxazaphosphorine toxicities.
It is a well-known fact that neoplasms are deleterious and reduce quality of life. Many alkylating cytotoxic agents, which cyclophoshpamide (CP) is a member, have been well documented to be effective in management of many human malignancies in order to improve quality of life and extend patients life span (Philip et al., 1961; Colvin, 1978; Friedman et al., 1979; Carter and Livingston, 1982). Despite its adverse effects, many clinicians have
continued to use CP either alone or in combination with other agents in cancer chemotherapy due to its efficacy.
Sodium-2-mecarptoethane sulfonate (MESNA), a sulfhydryl-containing agent has long been used in detoxifying and ameliorating specifically the urotoxic effects of CP and other oxazaphosphorines. However, its own side effects are equally disturbing and needs to be addressed (Reinhold-Keller et al., 1992).
Natural products have recently gained acceptance and have continued to gain grounds in therapeutics due to their acclaimed efficacy in management of many ailments with little or no side effects when used appropriately. They are also readily available depending on the region and geographical distribution. Many natural products have been widely reported to ameliorate at varying degrees the side effects of oxazophosphorines e.g. cyclophosphamide and ifosphamide (Łukasz and Piotr, 2012).
Many researchers have shown that Vernonia amygdalina Del. and Ocimum gratissimum Linn. are capable of detoxifying the body owing to their antioxidant properties thereby protecting the essential organs like the liver, kidney, heart, etc. (Owolabi et al., 2008; Arhoghro et al., 2009; Asuquo et al., 2010).
This study investigated the protective roles V. amygdalina and O. gratissimum in CP-induced urotoxicity and myelosupression in rats.
Pharmacology of cyclophosphamide
Figure 1: Chemical structure of Cyclophosphamide
Cyclophosphamide, [2-[bis(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxzaphosphorine-2- oxide] is one of the most effective and widely used cytotoxic antitumor agents. Cyclophosphamide (CP) and its structural isomer ifosfamide (N, 3-(bis(2-chloroethyl)- tetradydro-2H-1,3,2-oxazaphosphorin-2-amine 2-oxide, (IFEX or HOLOXAN), belong to oxazaphosphorine DNA alkylating agents widely used in cancer chemotherapy (Gilman, 1963).
Cyclophosphamide (CP) is a cytotoxic alkylating drug with a high therapeutic index and broad spectrum of activity against a variety of cancers (Fleming, 1997; Baumann and Preiss, 2001). It is often employed in the treatment of a variety of human malignancies despite its serious adverse effects which include: myelosuppression, haemorrhagic cystitis, nausea, vomiting, alopaecia, nephrotoxicity, immunotoxicity, mutagenicity, carcinogenicity and teratogenicity (Mirkes 1985; Fleming, 1997).
Cyclophosphamide was found to have antitumor activity in early 1940s (Gilman, 1963) and was later introduced to clinical cancer chemotherapy. Cyclophosphamide is often used in combination with other antineoplastic agents like methotrexate and fluorouracil to achieve higher efficacy in management of wide range of solid tumours and blood disorders (Zhang et al., 2006).
In non-neoplastic disorders, it has been found useful in organ transplantation procedure (Zinke et al., 1977) and treatment of some auto-immune diseases (Lares et al., 1971) due to its differential immunosuppressive effects (Schwartz and Grindey, 1973). Although the myelosuppressive effects of cyclophosphamide may be useful clinically, it is however completely undesirable in patients receiving cyclophosphamide regimen in management of neoplastic disorders.
Pharmacodynamics/mechanism of action of CP
The mechanisms of action of oxazaphosphorines are similar. Like other alkylating agents, the mechanism of action of alkylating nitrogen mustard of CP is through the covalent bonding of highly reactive alkyl groups with nucleophilic groups of nucleic acid. Cyclophosphamide itself is devoid of alkylating activity and must first undergo metabolic activation catalysed by the hepatic cytochrome P450 (CYP) monoxygenase systems (Boddy and Yule, 2000). Following metabolic activation, bifunctional alkylating nitrogen mustards of CP is generated, which are capable of reacting with the nitrogen-7 atom of purine bases in DNA, especially when they are flanked by adjacent guanines (Kohn et al., 1987). At alkaline or neutral pH, the nitrogen mustard is converted to chemically reactive carbonium ion through imonium ion. Carbonium ion reacts with the N7 of the guanine residue in DNA to form a covalent linkage. The second arm in phosphoramide mustard can react with a second guanine moiety in an opposite DNA strand or in the same strand to form crosslinks (Springer et al., 1998). The O6 atom of guanine may also be a target for oxazaphosphorines (Friedman et al., 1999). The different intramolecular distance between the chloroethyl group in CP mustard results in a different range of cross-linked DNA. Despite a good understanding of nature of the chemical reactions between alkylating species and DNA molecules (Shulman-Roskes et al., 1998), the mechanisms linking adduct and crosslink formation with tumor cell death are not
fully identified. Cyclophosphamide and ifosphamide, as with all other alkylating agents, destroy tumor cells through apoptosis (programmed cell death) initiated by DNA damage, modulation of cell cycle and other antiproliferative effects (O'Connor et al., 1991; Bhatia et al., 1995; Crook et al., 1986). It is generally accepted that the main mechanism that results in cell death is inhibition of DNA replication, as the interlinked strands do not allow separation of the two strands (Schwartz, 1983; Schwartz and Waxman, 2001). Apoptosis is characterized by a cascade-like activation of intracellular cysteineproteases (i.e. caspases). Caspases pre-exist as zymogens that are activated by proteolytic cleavage by other caspases or by autocatalysis. Distinct caspase cascades are involved in receptor-mediated or chemical- induced apoptosis (Sun et al., 1999). Drug-induced apoptosis is always mediated by the mitochondrial pathway leading to activation of the initiator caspase-9, which in turn activates the effector caspases-3 and caspase-7. Because CP can damage DNA during any phase of the cell cycle and its cytotoxicity is independent of the cell-cycle (Bruce et al., 1966), it is not cell cycle phase specific.
Pharmacokinetic profile of cyclophosphamide
Absorption: Considerable inter-personal variability exists in pharmacokinetics of CP. This is markedly influenced by route of administration, duration of treatment, patient age, coadministration with other drugs, genetic factors, functional status of the liver and the kidneys (Zhang et al., 2006).
As a monohydrate, CP is readily soluble in water, saline or alcohol. Oral administration is convenient and is well absorbed. The peak concentration appears 1hour following oral drug administration. The oral bioavailability is between 85 – 100% (Wagner and Fenneberg, 1984).
A fraction of the drug undergoes first pass metabolism in the liver and gut. At higher doses (0.7 g/m2), CP has about 87.7% oral bioavailability (Matthias et al., 1984).
Distribution: After oral or intravenous administration, CP is readily distributed throughout the body. About 30% binds to plasma protein (Moore, 1991). Its metabolite 4- hydroxycyclophosphamide (4-OH-CP) has higher plasma protein binding (approximately 67%) (Moore, 1991). CP is not structurally modified by blood plasma. The volume of distribution (Vd) of CP is increased in obese patients, leading to an increased elimination half-life (t1/2) of CP (Powis et al., 1987). Several studies suggested that CP entered into cerebrospinal fluid through blood-brain barrier (BBB) with varying cerebrospinal fluid to plasma ratios from 0.2 to 4% (Neuwelt et al., 1984; Yule et al., 1995; Hommes et al., 1983). The active metabolites of CP have limited penetration into the brain due to their increased polarity and higher plasma protein binding (Hommes et al., 1983). This may contribute to the lack of neurotoxicity associated with the intravenous administration of CP unlike its structural isomer, ifosphamide which has more extensive distribution with lower plasma protein binding thus making it more neurotoxic than CP (Zhang et al., 2006)
Transport: CP and its active metabolites are extensively bound by erythrocytes, which may serve as transporters of activated CP metabolites (Highley et al., 1996; Momerency et al., 1996; Dumez et al., 2004). The protoxic metabolite of CP, 4-hydroxy-CP, is trapped intracellular and transported to tumor tissues. There are increasing data on the transport of CP and its metabolites across cellular membrane. It is highly hydrophilic and do not diffuse readily through the lipid bilayer of cells (Highley et al., 1997). Similarly, the phosphoramide mustard, the cytotoxic but unstable metabolite of CP bears a negative charge with pKa of 4.5
- 4.8 at physiological pH and are thus relatively difficult to pass the cellular membrane (Zhang et al., 2006). 4-hydroxycyclophosphamide is the corresponding circulating metabolite that enters tumor cells to form ultimate cytotoxic phosphoramide mustard and the byproduct acrolein. It appears that 4-OH-CP and acrolein can readily cross the cell membrane by
passive diffusion in vitro. However, active transport cannot be excluded for CP and its metabolites based on their transport studies (Zhang et al., 2006).
Biotransformation/Metabolism. The initial activation of CP is achieved through 4- hydroxylation at C4 of oxazaphosphorine ring by hepatic CYP2B6, CYP3A4 and CYP2C9 to form 4-hydroxycyclophosphamide (4-OH-CP), which enters blood and is transported to tumor cells by erythrocytes (Colvin et al., 1976, Chang et al., 1993; Huang et al., 2000; Chen et al.,2004). The CYP2B6 is the major contributor (a mean of about 45% of total metabolism) for the activation of CP with the highest intrinsic clearance in vitro and in vivo, compared with 25% and 12% for CYP3A4 and CYP2C9, respectively (Roy et al., 1999; Huang et al., 2000; Chen et al., 2004). Other CYPs including CYP2A6, CYP2C8 and CYP2C19 also make a minor contribution to CP 4-hydroxylation (Chang et al., 1993). The 4- OH-CP so formed, is the major circulating metabolite of CP which is in equilibrium with its tautomer aldophosphamide that can decompose by β-elimination to form ultimate cytotoxic phosphoramide mustard (N,N-bis-2-(2-chloroethyl) phosphorodiamidic acid) and an equimolar amount of the byproduct acrolein (a highly electrophilic α,β-unsaturated aldehyde) (Hohorst et al., 1976; Fenselau et al., 1977; Colvin, 1999).
Acrolein is implicated in urotoxic effect of CP. It is detoxified by conjugation with glutathione (GSH). Alternatively, 4-hydroxycyclophosphamide is detoxified to form O- carboxyethylcyclophosphoramide mustard (CEPM, namely, carboxyphosphamide) primarily by aldehyde dehydrogenase (ALDH1A1) and, to much lesser extent, by ALDH3A1 and ALDH5A1 (Jarman, 1973; Domeyer and Sladek, 1980; Hipkens et al., 1981; Boddy et al., 1992). It is also oxidized by alcohol dehydrogenase (ADH) to form non-toxic 4- ketocyclophosphamide but at much lesser degree than CEPM (Hohorst et al., 1971; Lelieveld et al., 1976; Yule et al., 1995). Furthermore, 4-OH-CP undergoes reversible dehydration to form iminocyclophosphamide that is further conjugated with intracellular GSH, giving rise to
another non-toxic 4-glutathionylcyclophosphamide (GSCY), a substrate for multidrug resistance associated protein 2 (MRP2) exhibited by oxazaphosphorines (Hohorst et al., 1971). By contrast, the inactivation pathway of CP also involves minor (about10%) side chain oxidation (N-dechloroethylation) primarily by CYP3A4/3A5 and, to a minor extent, by CYP2B6 to generate 3-dechloroethyl-Ifosphamide and the neurotoxic and nephrotoxic byproduct chloroacetaldehyde (CAA) (Ren et al., 1997). The liver is the primary organ for the metabolism of CP through which the drug are activated and eliminated, but metabolism may occur in other sites, including the erythrocytes (Dockham et al., 1997), kidneys (Aleksa et al., 2005) and tumor itself (Schwartz et al., 2003). Various CYPs including CYP1A1, 2A6, 2B6, 2C8/9 and 3A4 are present in a variety of tumors, including those from the central nervous system, breast, colon, lung, ovarian, prostate and kidney, but their relative levels compared with normal tissue are less (Murray et al., 1995; Yu et al., 2001; Kivisto et al., 1995). Since CYP3A4 and CYP2B6 are the major enzymes for CP activation, their intratumoral level may be a useful predictive marker for the efficacy of CP treatment (Zhang et al., 2006).
Excretion: The CP is primarily (70 %) excreted in urine in forms of metabolites and to a less extent, in the feces (Boddy and Yule, 2000). However, only 10-20% is excreted unchanged in the urine (Fasola et al., 1991, Juma et al., 1979) and only 4% is excreted in the bile following CP administration (Dooley et al., 1982). CEPM is the dominant inactive metabolite of CP found in the urine, while 4-keto-CP is only a minor component in patient urine (<1%) (Hadidi et al., 1988). The renal clearance of CP in cancer patients was reported to be 15 – 44 ml/min (Busse et al., 1997).The reported renal clearance of CP metabolites including 4-OH-CP, dechloroethylifosfamide, keto-CP, and CEPM in cancer patients is 60.6
±9.0, 3.2 ± 1.0, 1.3 ± 0.8 and 7.0 ± 4.5 ml/min, respectively (Busse et al., 1997). These values are lower than the glomerular filtration rate (about125 ml/min). During high-dose
therapy of CP (100 mg/kg), the amount of CP excreted in urine is correlated with the urine flow, whereas this correlation does not exist during conventional dose therapy (500 mg/m2) (Busse et al., 1997). However, there was no correlation between the renal excretion of any metabolites of CP and urine flow during either conventional or high dose therapy (Busse and Kroemer 1997). Urine flow can have a significant effect of the renal clearance of CP that is substantially reabsorbed. The majority of CP elimination is by metabolic transformation with the metabolites recovered from the urine and feces. Thus, liver impairment may have an impact on pharmacokinetics and disposition of CP. However, such liver impairment only leads to less production of aldophosphamide; fewer adverse effects were observed in patients with liver dysfunction (Koren et al., 1992). Thus, it is not recommended to adjust the dosage of CP in patients with liver dysfunction (Zhang et al., 2006). A number of pharmacokinetic studies have been conducted with CP and the pharmacokinetic parameters reported. The t1/2 of CP ranges from 3.2 - 7.6 hours with total body clearance (CL) values of about 2.5 to 4.0 L/h/m2 (Boddy and Yule, 2000).
Pharmacokinetic variability: Large inter-patient variability in clinical response rate and toxicity has been observed in cancer patients treated with CP. This may be explained by differences in the pharmacokinetics of the agent observed in cancer patients (Yule et al., 1996; Ren et al., 1998). Inter-patient variability is often greater than intra-patient variability. Pharmacokinetic parameters of the parent drug vary less (coefficient of variation, 10-30%) than those for the elimination parameters (coefficient of variation, 14-64%) (Busse and Kroemer, 1997).
Inter-individual variations in the metabolism, transport, distribution and disposition of CP can be influenced by a number of factors associated with the drug (e.g. dosage, dosing
regimen, route of administration, and drug combination) and patients (e.g. age, gender, renal and hepatic function, and genetic factor).
Dosage: Dosage and dosing regimen are important factors affecting the pharmacokinetics of CP. There is increased interest in the use of high dose of CP in cancer chemotherapy. To increase chemotherapy efficacy against human cancer, it is desirable to increase dose intensity to the maximum tolerated dose. High dose oxazaphosphorine chemotherapy is assumed to result in improved antitumor activity due to increased generation of cytotoxic mustards (Zhang et al., 2006). Moderate and high dose CP, doxorubicin, and fluorouracil within the standard range result in greater disease-free and overall survival than the low dose regimen (Citron, 2004). Higher doses (>9.0 g/m2) of CP are usually administered intravenously in either 5% dextrose or 0.9% saline. Clinically, the drug is often given in a single dose over a period of up to 1 hour, repeated every 3 to 4 weeks. However, despite the use of myeloid growth factor and sulfhydryl compounds (e.g. MESNA and amifostine), myelosuppression continues to be a dose-limiting toxicity of oxazaphosphorines (Zhang et al., 2006). At higher doses used prior to marrow transplantation, the dose-limited toxicity is cardiac toxicity. Besides cardiac toxicity, hemorrhagic cystitis, water retention and hyponatremia are found in patients receiving high dose CP (Zhang et al., 2006). High-dose regimens have led to concerns over the existence of dose-dependent pharmacokinetics, with an increase in the production of inactive metabolites as the predominant activation pathway of metabolism is saturated.
The degree of renal excretion and inactive metabolite formation was increased at higher dose of CP, associated with a relative decrease in the formation of the active metabolite (Busse et al., 1997; Busse and Kroemer, 1997). Saturation kinetics at doses of 1 and 4 g/m2 of CP has been observed in cancer patients (Chen et al., 1995). Dosing schedule has significant effect on the pharmacokinetics of CP. In pediatric treatment or dose schedule given prior to bone
marrow transplantation, the total dose can be fractionated over several days. There is no obvious evidence that benefits can be achieved from the prolonged intravenous infusions of CP (Mouridsen et al., 1976).
Age: This is an important factor affecting the pharmacokinetics of CP. Elderly patients with non-small cell lung cancer demonstrated a doubled t1/2, because of an increased volume of distribution (Vd) when total body, renal and non-renal clearance remained unaltered (Lind et al., 1989). Pediatric patients are a specific group of individuals, because they have distinct physiological features from adults. The t1/2 of CP has been shown to be shorter than in adults (Yule et al., 1996). The differences in body surface area will lead to larger variability of CP doses, which makes comparison between pharmacokinetic parameters more difficult. Since Vd of CP approximates that of body water, the Vd in children is significantly lower than in adults.
Renal function: Since CP is primarily (70%) excreted in urine, renal function may play a role in the pharmacokinetic variability of CP. Early studies by Bramwell et al. (1979) and Juma et al. (1981) found that alterations in renal function did not significantly alter the pharmacokinetics of CP in patients, and did not result in any clinically-relevant changes in response rate and toxicity. However, a later study by Haubitz et al. (2002) indicated that the clearance of CP was decreased in patients with impaired renal function, thereby resulting in an increased systemic drug exposure. It appears that minor to moderate renal function impairment, insignificantly alters the clearance of CP or its alkylating metabolites and as such there is no necessity to adjust the doses of CP (Bramwell et al. 1979; Juma et al. 1981). However, terminal renal insufficiency may have a major impact on the renal excretion of CP and its metabolites. In particular, CP was removed by being taken into the dialysate in hemodialysis-dependent patients (Haubitz et al. 2002). Up to 25 % of administered CP dose (1.2 ± 0.4 g) was recovered in the dialysate and thus removed from the body during the
dialysis. The conclusion was that dialysis should not be initiated earlier than 12 hours after CP infusion, which can prevent the removal of drug in the early distribution phase (Haubitz et al. 2002). Only when the patients with terminal renal insufficiency are overdosed with CP, that the repetitive dialysis can help remove the drug from the body. Therefore, the severity of renal impairment has to be carefully assessed, dosage of CP should be accordingly adjusted in patients with renal dysfunction on hemodialysis and therapeutic drug monitoring should always be conducted in these patients (Zhang et al., 2006).
Liver function: Cyclophosphamide is extensively metabolized by hepatic CYP2B6, 2C9 and 3A4, indicating that liver impairment has a major impact on pharmacokinetics of CP. However, such liver impairment only leads to less formation of aldophosphamide; fewer adverse effects were observed in patients with liver dysfunction (Koren et al., 1992). Therefore, it is not recommended to adjust the dosage of CP in patients with liver dysfunction. Although biliary concentrations of CP are comparable to plasma concentrations, only a low fraction of CP (1.8 %) is found in stool (Dooley et al., 1982).
Disease status: The activation of CP was inhibited in tumor-bearing rats compared to healthy control (Sladek et al., 1978). The clearance of CP was found to reduce in children with Fanconi’s anemia, probably as a result of altered CYP oxidase-reductase cycling (Yule et al., 1999). Recently, the risk of recurrence of non-Hodgkin’s lymphoma in children is related to inadequate clearance of CP to active metabolites (Yule et al., 2004).
Genetic factor: Importantly, genetic factors may affect the pharmacokinetics of CP. There are increased studies on the role of genetic polymorphisms of genes encoding various proteins involved in the distribution, metabolism, and transport of CP in the pharmacokinetic variability and therapeutic outcomes (Zhang et al., 2006). Theoretically, polymorphisms of CYP3A4, CYP2B6, CYP2C9, ALDH1A1, ALDH3A1, GSTT1, GSTM1, GSTP1, and MRP2
may play a role in the disposition of CP, thus resulting in wide inter-patient variability in
exposure to CP and its active metabolites, with important clinical consequences in cancer chemotherapy (Wormhoudt et al., 1999; Desta et al., 2002; Rodrigues et al., 2002). Since CP undergo extensive CYP-catalyzed metabolism through which it is activated, deactivated and eliminated, drug interactions may arise due to modulation of the pharmacokinetics, in particular, when inhibition or induction of the relevant CYPs is implicated.
Therapeutic uses: CP is the most widely used alkylating agent in the treatment of hematological malignancies and a variety of solid tumors, including leukemia (Demirer et al., 1996, Rao, et al., 2005), breast cancer (Lippman et al.,1986; Levine, et al.,2005), lung cancer (Chrystal et al., 2004; Hobdy 2004), lymphomas (Escalon et al., 2005; Zinzani, 2005; Kasamon et al 2005), prostate cancer (Nicolini et al., 2004; Hellerstedt et al., 2003;), ovarian cancer (Morgan et al. 2001; Inoue et al., 1995; Nicoletto et al., 2004), and multiple myeloma (Dimopoulos et al., 2004; Lundin et al., 2003). This agent has similarly been used extensively for the treatment of diffuse proliferative glomerulonephritis in patients with lupus erythematosus affecting the kidneys (Hengstler et al., 1997). Although its role in the treatment of ovarian cancer and small-cell lung cancer is declining, CP continues to be used in treatment of breast cancer as a critical component of the CMF (CP, methotrexate, flourouracil), CEF (CP, epirubicin, and 5-fluorouracil), MVC (mitoxantrone, vinblastine, and CP) and DDC (docetaxel, doxorubicin and CP) regimen (Levine et al., 2005; Stewart et al., 2005). Higher doses of CP are used in the treatment prior to bone marrow transplantation for aplastic anemia, leukemia and other malignancies (Demirer et al., 1996, Rao, et al., 2005). Recently, there is accumulating evidence indicating the action of CP on the immune system. CP has modulating effects on both humoral and cell-mediated immunity (Zhang et al., 1993; Brodsky et al., 2002; Lacki et al., 1997) and thus beneficial effects are obtained when used as an immunosuppressive drug. CP also augmented the efficacy of antitumor immune
responses in animals and humans by depleting CD4+/CD25+ regulatory T cells and increasing T lymphocyte proliferation and T memory cells (Ikezawa et al 2005; Lutsiak et al., 2005). The immunostimulatory effect of CP is associated with the marked inhibition of inducible nitric oxide synthase (Loeffler et al., 2005). Furthermore, CP kills circulating endothelial progenitors that present as a marker of tumor angiogenesis (Mancuso et al., 2003), whereas 4-OH-CP readily destroys various hematopoietic progenitors cells including marrow stromal progenitors (Siena et al., 1985). These findings may provide a solid rationale for the use of CP as an immunosuppressive agent in the treatment of autoimmune diseases or an integral component in combination with other immunotherapy in cancer treatment.
A number of drug interactions with CP have been reported in humans. It seems that the underlying mechanism is inhibition of CYP enzymes for the drug interactions with allopurinol (Yule et al., 1996), chloramphenicol (Faber et al., 1975), sulphaphenazole (Faber et al., 1975), chlorpromazine (Yule et al., 2004), fluconazole (Yule et al., 1999), ranitidine (Alberts et al., 1991), and triethylenethiophosphoramide (THIOTEPA) (Anderson et al., 1996). Drug interactions have also been reported with dexamethasone (Yule et al., 1996), prednisolone (Faber et al., 1974), phenobarbitone (Jao et al., 1972), and phenytoin (Slattery et al., 1996) due to induction of CP metabolism. Phenytoin induces the N-dechloroethylation of the S-enantiomer of CP to a greater extent than that of the R-enantiomer (Williams et al., 1999). The clinical significance of these drug interactions is unclear. In addition, an altered toxicity profile of CP was observed when combined with paclitaxel in a schedule-dependent manner (Kennedy et al., 1998), but pharmacokinetic modulation cannot provide an explanation.
On the other hand, CP may alter the pharmacokinetics and pharmacodynamics of co- administered
drugs. Cyclophosphamide reduced digoxin absorption has been reported (Rodin and Jonhson, 1988). Triethylenethiophosphoramide inhibited the activation of CP and decreased the efficacy and toxicity of CP (Anderson et al., 1996), because of the mechanism-based inhibition of CYP2B6 (Rae et al., 2002). Triethylenethiophosphoramide is frequently given in conjunction with CP in high-dose chemotherapy regimens in preparative regimens before autologous bone-marrow and peripheral stem-cell transplantation. Therefore, the sequence and schedule of these two drugs should be critically considered and it has been recommended that these two agents should not be combined (Huitema et al., 2000). Furthermore, the clearance of doxorubicin was significantly reduced (50%) when in combination with CP (Zhang et al., 2006). The combination of CP and doxorubicin also caused a 10% decrease in clearance of etoposide, but this is of little clinical significance (Zhang et al., 2006). Cyclophosphamide is often combined with doxorubicin and etoposide in the treatment of breast cancer. Therefore, proper therapeutic drug monitoring is needed when CP is used in combination with doxorubicin and etoposide (Zhang et al., 2006).
Mechanism of CP toxicity
Cyclophosphamide is primarily activated in the liver by CYP3A4, CYP2C9 and CYP2B6 followed by erythrocyte- mediated transport of the activated metabolites to the tumor tissue via blood circulation (Zhang et al., 2006). Cyclophosphamide is considered a prodrug as it requires activation by mixed function oxidase enzyme in the liver (hepatic cytochrome P450 monoxygenase system) to yield the main active metabolite, phosphoramide mustard, and the by-product acrolein (Foley et al., 1961; Struck et al., 1975). However, these activated metabolites, phosphoramide mustard and acrolein also gain entry into normal tissues, where they may induce host toxicity. The resultant phosphoramide mustard is a bifunctional alkylator of DNA and the ultimate cytotoxic metabolite of CP (Struck et al., 1975). The
alkylation involves generation of the intermediate phosphoramide aziridinium ion through an intramolecular nucleophilic attack (cyclization reaction) of the nitrogen on the β-carbon of a chloroethyl chain (Ludeman, 1999). Cellular thiols (e.g., GSH) and other nucleophiles react rapidly with phosphoramide aziridinium ions, resulting in thioether products (Gamcsik et al., 1999). Carboxyethylcyclophosphoramide mustard (CEPM) is one of the major chemically stable metabolites of CP, which are easily detected in patient plasma and urine (Joqueviel et al., 1998). However, acrolein is a highly reactive aldehyde that covalently binds to cellular macromolecules and subsequently disrupts the function and causes organ toxicity (Brock et al., 1979; Kehrer and Biswal, 2000). It is detoxified by conjugation with GSH through glutathione-s-transferases (GSTs) in hepatocytes (Gurtoo et al., 1981a) and this may cause intracellular GSH depletion and injuries of the hepatocytes and urothelium (DeLeve et al., 1996). Reaction of GSH with acrolein is via nucleophilic addition at the β-carbon atom, generating stable thioether compounds for elimination (Ramu et al., 1995).
Outcomes of CP Toxicity
The usual dose-limiting toxicity is myelosuppression (Bramwell et al., 1987). Since CP is a non-specific alkylating agent, severe host toxicity is inevitable. The urological side effects of CP are major limiting factor for its use (Brock and Pohl, 1986; Honjo et al., 1988). These side effects include transient irritative voiding symptoms including dypsia, hemorrhagic cystitis, bladder fibrosis, necrosis, con-tracture and vesicometral flux (Levine and Richie, 1989). Several reports have also linked cases of lung toxicity to this medication (Patel et al., 1984). Similarly, several studies have demonstrated that bladder inflammation induced by CP in rats and mice increased transcript and protein expression in the urinary bladder of several cytokines including IL-6 (Malley and Vizzard, 2002). At higher doses used prior to marrow transplantation, the dose-limited toxicity is cardiac toxicity (Peters et al., 1989). Besides cardiac toxicity, hemorrhagic cystitis, water retention and hyponatremia are found in patients
receiving high dose CP. Acrolein is the causal agent of hemorrhagic cystitis. Using MESNA (sodium-2-mercaptoethanesulfonate) can reduce the incidence of hemorrhagic cystitis (Brock and Pohl 1986). A direct effect of CP on the renal tubules leads to excess water retention. This can be managed by hydration with isotonic fluids (Bode et al., 1980). In women receiving CP, methotrexate and fluorouracil for treatment of breast cancer, severe thromboembolic events have been reported (Pritchard et al., 1997). Elevation of serum level of aminotransferases in patients treated with CP has also been reported. This CP-induced liver injury results from metabolites of CP, especially acrolein and is mainly dose-dependent (Honjo et al., 1988). Metabolites of CP have been demonstrated to be teratogens and carcinogens in animals. Malformations have been associated with first trimester exposure to CP (Zemlickis et al. 1993). It is reported that CP as a single chemotherapeutic agent, induces leukemia in human (Haas et al., 1987). The mechanisms are unknown, but this may be associated with the cytogenetic toxicity of CP (Au et al., 1980). Patients with breast cancer and inheritance of a combined gene deletion of GSTM1 and GSTT1 might bear an increased risk to develop a secondary CP-induced hematological neoplasia (Haase et al., 2002). Cyclophosphamide treatment also leads to lung injury and cardiac toxicity, which are mediated by the reactive oxygen species (ROS) and lipid peroxide formation (Patel, 1987; Sulkowska et al., 1998). Cyclophosphamide treatment not only induces lipid peroxidation (LPO) but also suppresses the tissue and serum level of reduced glutathione (GSH), glutathione peroxidase (GP), glutathione reductase, superoxide dismutase (SOD) and catalase (CAT) activity (Sulkowska et al., 1998; Kaya et al., 1999)..