*2.2.1 Substrates*

*Advances in Precision Medicine Oncology*

activation of the p-38/MAPK pathway [50].

protein-1 (YB-1) [37, 38], activator protein-1 (AP-1) [39], and hypoxia-inducible factor-1 (HIF-1) [38, 40]. The activities of these transcription factors have been linked to various signal transduction pathways, particularly the two major cell survival signaling cascades i.e. (1) the mitogen-activated protein kinase (MAPK) [37, 41], and (2) the phosphatidylinositol 3-kinase (PI3K) pathways [42, 43]. It has been shown that hyperactivation of either MAPK/ERK1/2 or PI3K/Akt/NF-ᴋB signaling pathways results in overexpression of P-gp in doxorubicin-resistant cells such as lung, breast and ovarian cancer cells [44–49]. An up-regulation of P-gp expression in vincristine-resistant human gastric cancer cells was associated with

After activation and translocation into nucleus, transcription factors such as NF-kB and YB-1 (Y-box binding protein) bind to MDR1 promoter region, leading to initiation of MDR1 transcription. Increase in YB-1 nuclear activity is related to P-gp-mediated development of MDR in several cancers including breast cancer, lung cancer, ovarian cancer, colorectal cancer, prostate cancer and osteosarcoma [38]. In response to cell stress such as hyperthermia, viral infection and chemical assault, the survival Akt and MAPKs signaling would be activated, and subsequently increase YB-1 expression and translocation into nucleus for its MDR1-transcription activity [51]. Doxorubicin is a known P-gp inducer in various cancer cells. Doxorubicin up-regulates MDR1 gene expression via the MAPK/ERK1/2 signaling that linked to activation of YB-1 in B-cell lymphoma [37]. Moreover, upregulation of P-gp has been reported after prolonged exposure to various functional unrelated compounds, leading to the loss of drug efficacy and safety [52]. Examples of the known P-gp inducers include anticancer (e.g., cisplatin, doxorubicin, etoposide vinblastine), antidepressants (e.g., trazodone, St. John's Wort), anticonvulsants (e.g., carbamazepine, phenytoin), anti-HIV (e.g., saquinavir, indinarvir, tenofovir), immunosuppressants (e.g., cyclosporine, tacrolimus), steroids (e.g., dexamethasone) [52–54]. It is worth noting that certain CYP450 inducers such as rifampin and St. John's Wort are able to up-regulate P-gp expression, possible sharing through the PXR regulation [55, 56]. Prolonged exposure to rifampin and St. John's Wort in human led to increased intestinal P-gp level, and increased digoxin absorption [57, 58]. Since, P-gp-mediated MDR in cancer is largely due to up-regulation of P-gp expression, better understanding of the signaling proteins and transcription factors will provide

a promising targets in overcoming MDR for anticancer chemotherapy.

Overexpression of P-gp has been strongly correlated with chemo-resistance and cancer relapses in several cancer patients such as breast cancer, adult acute myeloid leukemia, pheochromocytoma patients, leading to poor prognosis from therapeutic failure in patients receiving chemotherapy [1, 59–62]. Accordingly, P-gp is intrinsically expressed in various cancer types, particularly those derived from tissues with high basal MDR1 expression levels such as colon, kidney and liver tissues. Being a transmembrane efflux pump, P-gp serves as a cellular defense mechanism against drug assault by limiting intracellular drug accumulation up to toxic threshold level. Regarding this, the susceptibility of cancer to anticancer drugs being P-gp substrate varies, depending on intrinsic expressed P-gp levels. Certain types of cancers may be classified as poor responder showing their unresponsiveness to chemotherapy regimens containing P-gp substrates. For example, prostate cancer appears to be better responder to chemotherapy, as compared to colorectal or renal cancers [63, 64]. Moreover, some cancers such as leukemia, lymphoma and breast cancer having low levels of intrinsic P-gp expression, and thus initially respond well to

*2.1.3 P-gp and multi-drug resistance in cancer*

**192**

Human ABC efflux transporters demonstrate their broad substrate specificity toward structurally diverse lipophilic compounds. Most of their substrates are weakly amphipathic and hydrophobic planar structure with aromatic ring and positively charged nitrogen atom [52, 54, 67, 68]. Examples of P-gp substrates are anticancer drugs (vinca alkaloids, anthracyclines, and epipodophyllotoxins), cardiovascular drugs (e.g., digoxin, quinidine, talinolol, diltiazem, losartan, verapamil), anti-microbial agents (e.g., doxycycline, erythromycin, itraconazole, rifampin), anti-viral drugs (e.g., indinavir), anticonvulsants (e.g., phenytoin), acid blockers (e.g., cimetidine), immunosuppressants (e.g., cyclosporine, tacrolimus), steroids (e.g., aldosterone, cortisol, dexamethasone), opioids (loperamide, morphine).


#### **Table 1.**

*Selected substrates, inducers and modulators of P-gp.*

#### *2.2.2 Modulators*

Modulators suppress P-gp activity through either (1) direct inhibition of P-gp function by either competitive or non-competitive inhibitors; or (2) suppression of P-gp expression levels by interferences with either transcription, translation/ post-translation, and degradation processes.

#### *2.2.2.1 Direct inhibition of functionally active P-gp*

The direct inhibition of active P-gp can be attributed to the interaction between chemicals and P-gp at either TMB or NBD [67–69]. Any compound such as tyrosine Kinase Inhibitors interferes with ATP binding or hydrolysis in NBD site can reduce P-gp transport action [70]. Chemicals identified as small molecule P-gp inhibitors such as amiodarone, diltiazem, verapamil bind to substrate binding sites or allosteric sites in TMB, resulting in interference on substrate binding and transport. It has been reported that certain compounds such as cyclosporine A could exert their inhibitory action by interfering with substrate recognition and ATP hydrolysis [8, 67–69]. It is not surprising that these TMB type inhibitors and substrates share many common molecular features such as hydrophobic planar structure. In addition, due to the diversity in chemical structure of P-gp inhibitors, establishment of the structure activity relationship (SAR) of P-gp inhibitors is very challenging. The structure pattern of the inhibitors contains planar rings and basic nitrogen atom within an extended side chain of the aromatic ring. The presence of tertiary amino groups, in comparison with primary and secondary amine, increases the anti-MDR potency considerably. Furthermore, the presence of nitrogen atom in non-aromatic ring apparently increases inhibitory action of the compounds [71]. Examples of P-gp inhibitors are calcium channel blockers (verapamil, diltiazem), and various phytochemicals such as flavonoid and steroidal compounds (e.g., quercetin, resveratrol), indole alkaloids and polycyclic compounds (e.g., capsaicin, piperine, rhinacanthin C) [66, 72–74].

Ideally, the P-gp inhibitors should be potent and selective to P-gp function at target cells/tissues, with no systemic side effects. To date, there are four generations of small molecule inhibitors. The first generation inhibitors are known drug substrates of the ABC transporters such as verapamil, cyclosporine A, tamoxifen and quinidine [75]. They were not specifically designed to be P-gp inhibitors, and could not display good clinical outcomes in their MDR reversal activity. The clinical disappointment could be due to their weak inhibitory potency against the MDR transporters including P-gp, and their pharmacokinetic interactions with chemotherapeutic agents, leading to the need of high doses and intolerable adverse effects [1, 76]. Next, the second generation inhibitors such as valspodar (cyclosporine A derivative) were developed, based on structure activity relationships of the first generation compounds, in order to improve potency, specificity, and to reduce systemic toxicity. Although this group of inhibitors demonstrated their improvement in inhibitory potency, their clinical outcomes were still unsatisfied due to their pharmacokinetic interaction with the anti-cancer drugs via inhibition of cytochrome P450, and their severe toxicity [75, 77]. Subsequently, the third generation P-gp inhibitors such as elacridar, tariquidar and zosuquidar were developed in order to address the limitations of the second generation compounds. These inhibitors elicit no effect on CYP P450 metabolism, therefore they are unlikely to affect the plasma concentrations of anti-cancer drugs. They were also more potent and selective P-gp inhibitors, effectively working in nanomolar concentration range. However, the potent P-gp inhibitor tariquidar can be either substrate or inhibitor of P-gp depending on its given dose [78]. To date, the clinical efficacy for MDR reversal of this generation has yet completely satisfied, its effectiveness possibly also depends on given dosage and intrinsic tumor properties.

**195**

*Overcoming P-Glycoprotein-Mediated Doxorubicin Resistance*

transporter-independent MDR mechanisms [75, 82–86].

*2.2.2.2 Suppressor of P-gp expression*

In addition to small molecule inhibitors, monoclonal antibodies can be another alternative approach in inhibiting P-gp activity. Theoretically, any agents that specifically affect lipid-protein interactions or protein structure of targeted P-gp can be developed into P-gp inhibitor. Typically, monoclonal antibody can be developed to specifically recognize and bind to its target protein, leading to inhibition of changes in protein conformation. Regarding this, human P-gp-specific antibodies UIC2, MRK-16 and 4E3 reacted specifically to the extracellular loop of both halves of P-gp, and disabled P-gp transport activity [87]. Consequently, treatment cancer cells with these antibodies resulted in increased concentrations of anticancer drugs (e.g., vincristine, actinomycin D, doxorubicin, paclitaxel) within the cells, and improve drug effectiveness [87–91]. In athymic mice model, MRK16 was demonstrated its ability to significantly reduce tumor mass [92]. Further clinical studies of human P-gp-specific antibodies are needed to conduct in terms of safety and efficacy.

In addition to direct inhibition, reduction of P-gp activity can arise from decrease of protein expression at plasma membrane. Interference on transcription and translation of MDR1 gene, resulting in reduction of P-gp expression, can be another approach to overcome MDR in cancer. Several innovative tools targeting at MDR transcription or mRNA including small molecules, antisense oligonucleotides, hammerhead ribozymes and RNA interference strategies have been employed.

Currently, phytochemicals or natural compounds with MDR reversal activity have been subject of interest in searching for new effective chemo-sensitizer against cancer. This group of inhibitors obtained from natural sources is classified as the fourth generation inhibitor. Numerous phytochemical researches on pharmacological activities and pharmacokinetics have revealed that plant-based compounds elicit a broad spectrum of pharmacological actions such as anti-cancer, anti-oxidant, anti-microbial, anti-inflammation, etc. In addition, these plant-based compounds, depending upon its molecular structure, may interfere with P-gp and metabolizing enzymes, leading to the concerning issues in drug bioavailability and pharmacokinetic drug interactions. The advantages of the fourth generation inhibitors in part rely on their natural origin with long history of uses in dietary or health supplements and in traditional medicine. It may be able to presume that this group of inhibitors derived from known edible products possessed less toxicity and more tolerable than those of the previous generation compounds. Evidently, even vegetables (e.g., bitter melon), spices (e.g., black pepper, turmeric) or fruits (e.g., orange, grapefruit) also contain substances that could inhibit P-gp and other efflux transporters in the ABC superfamily [72–75, 77, 79–82]. Their competitive inhibition against the efflux transporters enhance cytotoxicity of anticancer drugs such as doxorubicin and vinblastine, leading to potential MDR reversal in various cancer cells. However, the inhibitory potency of these plant-based compounds against P-gp activity might be low. Their IC50 values obtained from the in vitro cell culture models appear to be in micromolar range. Thus, this group of inhibitors is unlikely a good MDR reversing agent through direct P-gp inhibition at MDR cancer cells in clinical setting. In addition, the interference of P-gp activity of these compounds in pharmacokinetic aspect may influence on P-gp-related ADME and bioavailability of chemotherapeutic drugs that concomitantly given. Nevertheless, the opportunities of further development into effective chemosensitizers cannot be excluded. Better understanding of QSAR may enable to facilitate chemical modification of these identified plant-based P-gp inhibitors to generate more potent and high selective P-gp inhibitors. Furthermore, several plant-based compounds (e.g, curcumin, resveratrol, quercetin) have been demonstrated their potential in down-regulation of P-gp and other key regulators in

*DOI: http://dx.doi.org/10.5772/intechopen.95553*

#### *Overcoming P-Glycoprotein-Mediated Doxorubicin Resistance DOI: http://dx.doi.org/10.5772/intechopen.95553*

*Advances in Precision Medicine Oncology*

post-translation, and degradation processes.

*2.2.2.1 Direct inhibition of functionally active P-gp*

Modulators suppress P-gp activity through either (1) direct inhibition of P-gp function by either competitive or non-competitive inhibitors; or (2) suppression of P-gp expression levels by interferences with either transcription, translation/

The direct inhibition of active P-gp can be attributed to the interaction between chemicals and P-gp at either TMB or NBD [67–69]. Any compound such as tyrosine Kinase Inhibitors interferes with ATP binding or hydrolysis in NBD site can reduce P-gp transport action [70]. Chemicals identified as small molecule P-gp inhibitors such as amiodarone, diltiazem, verapamil bind to substrate binding sites or allosteric sites in TMB, resulting in interference on substrate binding and transport. It has been reported that certain compounds such as cyclosporine A could exert their inhibitory action by interfering with substrate recognition and ATP hydrolysis [8, 67–69]. It is not surprising that these TMB type inhibitors and substrates share many common molecular features such as hydrophobic planar structure. In addition, due to the diversity in chemical structure of P-gp inhibitors, establishment of the structure activity relationship (SAR) of P-gp inhibitors is very challenging. The structure pattern of the inhibitors contains planar rings and basic nitrogen atom within an extended side chain of the aromatic ring. The presence of tertiary amino groups, in comparison with primary and secondary amine, increases the anti-MDR potency considerably. Furthermore, the presence of nitrogen atom in non-aromatic ring apparently increases inhibitory action of the compounds [71]. Examples of P-gp inhibitors are calcium channel blockers (verapamil, diltiazem), and various phytochemicals such as flavonoid and steroidal compounds (e.g., quercetin, resveratrol), indole alkaloids and

polycyclic compounds (e.g., capsaicin, piperine, rhinacanthin C) [66, 72–74].

also depends on given dosage and intrinsic tumor properties.

Ideally, the P-gp inhibitors should be potent and selective to P-gp function at target cells/tissues, with no systemic side effects. To date, there are four generations of small molecule inhibitors. The first generation inhibitors are known drug substrates of the ABC transporters such as verapamil, cyclosporine A, tamoxifen and quinidine [75]. They were not specifically designed to be P-gp inhibitors, and could not display good clinical outcomes in their MDR reversal activity. The clinical disappointment could be due to their weak inhibitory potency against the MDR transporters including P-gp, and their pharmacokinetic interactions with chemotherapeutic agents, leading to the need of high doses and intolerable adverse effects [1, 76]. Next, the second generation inhibitors such as valspodar (cyclosporine A derivative) were developed, based on structure activity relationships of the first generation compounds, in order to improve potency, specificity, and to reduce systemic toxicity. Although this group of inhibitors demonstrated their improvement in inhibitory potency, their clinical outcomes were still unsatisfied due to their pharmacokinetic interaction with the anti-cancer drugs via inhibition of cytochrome P450, and their severe toxicity [75, 77]. Subsequently, the third generation P-gp inhibitors such as elacridar, tariquidar and zosuquidar were developed in order to address the limitations of the second generation compounds. These inhibitors elicit no effect on CYP P450 metabolism, therefore they are unlikely to affect the plasma concentrations of anti-cancer drugs. They were also more potent and selective P-gp inhibitors, effectively working in nanomolar concentration range. However, the potent P-gp inhibitor tariquidar can be either substrate or inhibitor of P-gp depending on its given dose [78]. To date, the clinical efficacy for MDR reversal of this generation has yet completely satisfied, its effectiveness possibly

*2.2.2 Modulators*

**194**

Currently, phytochemicals or natural compounds with MDR reversal activity have been subject of interest in searching for new effective chemo-sensitizer against cancer. This group of inhibitors obtained from natural sources is classified as the fourth generation inhibitor. Numerous phytochemical researches on pharmacological activities and pharmacokinetics have revealed that plant-based compounds elicit a broad spectrum of pharmacological actions such as anti-cancer, anti-oxidant, anti-microbial, anti-inflammation, etc. In addition, these plant-based compounds, depending upon its molecular structure, may interfere with P-gp and metabolizing enzymes, leading to the concerning issues in drug bioavailability and pharmacokinetic drug interactions. The advantages of the fourth generation inhibitors in part rely on their natural origin with long history of uses in dietary or health supplements and in traditional medicine. It may be able to presume that this group of inhibitors derived from known edible products possessed less toxicity and more tolerable than those of the previous generation compounds. Evidently, even vegetables (e.g., bitter melon), spices (e.g., black pepper, turmeric) or fruits (e.g., orange, grapefruit) also contain substances that could inhibit P-gp and other efflux transporters in the ABC superfamily [72–75, 77, 79–82]. Their competitive inhibition against the efflux transporters enhance cytotoxicity of anticancer drugs such as doxorubicin and vinblastine, leading to potential MDR reversal in various cancer cells. However, the inhibitory potency of these plant-based compounds against P-gp activity might be low. Their IC50 values obtained from the in vitro cell culture models appear to be in micromolar range. Thus, this group of inhibitors is unlikely a good MDR reversing agent through direct P-gp inhibition at MDR cancer cells in clinical setting. In addition, the interference of P-gp activity of these compounds in pharmacokinetic aspect may influence on P-gp-related ADME and bioavailability of chemotherapeutic drugs that concomitantly given. Nevertheless, the opportunities of further development into effective chemosensitizers cannot be excluded. Better understanding of QSAR may enable to facilitate chemical modification of these identified plant-based P-gp inhibitors to generate more potent and high selective P-gp inhibitors. Furthermore, several plant-based compounds (e.g, curcumin, resveratrol, quercetin) have been demonstrated their potential in down-regulation of P-gp and other key regulators in transporter-independent MDR mechanisms [75, 82–86].

In addition to small molecule inhibitors, monoclonal antibodies can be another alternative approach in inhibiting P-gp activity. Theoretically, any agents that specifically affect lipid-protein interactions or protein structure of targeted P-gp can be developed into P-gp inhibitor. Typically, monoclonal antibody can be developed to specifically recognize and bind to its target protein, leading to inhibition of changes in protein conformation. Regarding this, human P-gp-specific antibodies UIC2, MRK-16 and 4E3 reacted specifically to the extracellular loop of both halves of P-gp, and disabled P-gp transport activity [87]. Consequently, treatment cancer cells with these antibodies resulted in increased concentrations of anticancer drugs (e.g., vincristine, actinomycin D, doxorubicin, paclitaxel) within the cells, and improve drug effectiveness [87–91]. In athymic mice model, MRK16 was demonstrated its ability to significantly reduce tumor mass [92]. Further clinical studies of human P-gp-specific antibodies are needed to conduct in terms of safety and efficacy.

#### *2.2.2.2 Suppressor of P-gp expression*

In addition to direct inhibition, reduction of P-gp activity can arise from decrease of protein expression at plasma membrane. Interference on transcription and translation of MDR1 gene, resulting in reduction of P-gp expression, can be another approach to overcome MDR in cancer. Several innovative tools targeting at MDR transcription or mRNA including small molecules, antisense oligonucleotides, hammerhead ribozymes and RNA interference strategies have been employed.

#### *2.2.2.2.1 MicroRNA and RNA interference (RNAi) technologies*

Application of microRNA and RNAi technologies with either small-interfering RNA (siRNA) or small hairpin RNA (shRNA) to specific silence MDR1 expression in cancer cells with MDR phenotype has been demonstrated their effectiveness in down-regulation of MDR1 and P gp expression with paralleled increases drug accumulation and improved sensitivity to treatment. MicroRNAs (miRNAs) are small non-coding RNA molecules that can inhibit ABCB1 mRNA translation processes [93, 94]. A number of miRNAs have been studied on their ability to down-regulate P-gp expression and restore cell sensitivity to P-gp drug substrates in drug resistant cells [34]. For example, miRNA-4539 could increase doxorubicin-mediated cell death in MDA-MB-231 breast cancer cells [93, 94].

The RNAi technologies involve either transient gene-silencing by siRNA or stable inhibition by MDR1 shRNA-transfected on plasmid DNA of MDR cancer cells. Treatment with siRNA against MDR1 increases drug-mediated cytotoxicity in various MDR cancer cells such as paclitaxel in MDR1 ovarian cancer cells and doxorubicin in doxorubicin-resistant breast cancer cells [95]. In addition, siRNA was able to significant reduced size of doxorubicin-resistant xenograft in a mouse model [96]. MDR1 ShRNA transfected in taxol-resistant human ovarian cancer cell line A2780 effectively down-regulated P-gp expression, and enhanced paclitaxelmediated toxicity in this cells [97].

Selective suppression of P-gp/MDR1 expression with either microRNA or RNAi technologies offers the novel approach to specifically combat P-gp-based MDR in cancer, and re-sensitize the MDR cells to chemotherapeutic agents. However, for their therapeutic applications, there are several challenges required especially the effective miR/RNAi delivery to target cancer cells, design of expression vectors for shRNA, systemic stability and degradation, and safety of patients.

#### *2.2.2.2.2 Small molecules as P-gp down-regulator*

Numerous small molecules particularly those in the fourth generation of P-gp inhibitors such as curcumin, ginsenoside, quercetin and resveratrol have been demonstrated their ability to reduce P-gp function in the MDR cancer cells via down-regulation of P-gp expression [83–85]. By targeting at the signaling pathways related to transcription process of MDR1, several plant-based compounds suppress P-gp expression in the resistance cells and improve chemo-sensitivity to anticancer drugs. For instance, the P-gp modulating effect of asiatic acid, ginsenoside, isoquinoline alkaloids (e.g. cepharantine, tetrandine) resulted from their blockade of MAPK/ERK1/2 or PI3K/Akt pathways in MDR cancer cells [86, 98–101]. Another isoquinoline alkaloid berberine inhibited P-gp expression and enhanced doxorubicin-mediated toxicity in MCF-7 cells through down-regulation of AMPK-HIF1α signaling cascade [102]. Anti-MDR property of natural curcuminoids (e.g., curcumin, bisdemethoxycurcumin) involved with inhibition of human MDR1 gene expression in MDR cervical carcinoma KB-V1 cells [103]. In addition, certain compounds such as a natural marine product Et743 inhibit MDR1 transcription via blocking its promoter activation [104].

#### **3. Doxorubicin and P-gp**

Doxorubicin is one of the most effective cytotoxic anticancer drugs. This drug has been used for combating various types of cancers such as cancers of breast, ovary, prostate, stomach, thyroid; small cell cancer of lung; squamous cell cancer of

**197**

transporters.

pharmacological actions.

*Overcoming P-Glycoprotein-Mediated Doxorubicin Resistance*

head and neck; multiple myeloma; Hodgkin's disease; lymphomas; acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML). Unfortunately, the uses of doxorubicin can be limited because of its dose-related toxicity (e.g., nausea, vomiting, hair loss, leucopenia, cardiomyopathy, heart failure) and high MDR incidence [105, 106]. Despite the good clinical therapeutic responses are seen in patients receiving doxorubicin in the earliest stage of treatment, multi-drug resistance may

One of the major mechanisms responsible for doxorubicin-induced MDR is upregulation of MDR1/P-gp expression. Doxorubicin is an anthracycline derivative with a four-membered ring system containing an anthraquinone chromophore, and an aminoglycoside (**Figure 1**). This molecular structure accommodates its interaction with major MDR efflux transporters in the ABC superfamily proteins. It has been well established that doxorubicin and other anthracycline derivatives are P-gp substrates with ability to up-regulate P-gp/MDR1 expression after repeated exposure in various cancer cells such as breast and lung cancers as well as in vivo and in clinical settings [66, 107, 108]. For instance, lung perfusion with doxorubicin resulted in an increase of MDR1 RNA in patients with sarcoma pulmonary metastases [18]. The P-gp-overexpressed cancer cells would have intracellular doxorubicin concentration below its effective threshold level. Consequently, cancer cells increasingly survive from doxorubicin-mediated cytotoxity. In this circumstance, titrating dose up to overcome MDR may not enable to achieve a successful outcome due to dose-limiting toxicity. Because the adverse effects of doxorubicin and other anti-cancer drugs are mostly concentration-dependent, increasing doses can produce higher degree of severity and unendurable adverse events, leading to patient's intolerability and even fatal outcome. Addition of other cytotoxic drugs into doxorubicin-based regimens may not also enable to obtain a chemotherapeutic success, if those drugs are also substrates of the MDR

Generally, clinical efficacy of doxorubicin depends on its pharmacokinetics after systemic exposure influencing (1) the therapeutic concentration at target organs, and (2) the homogeneity of drug distribution in the cancerous tissues particularly solid tumor. In addition, it is very critical that doxorubicin accumulates within the targeted cancer cells at the level greater than its cytotoxic threshold to elicit its

Doxorubicin is poorly absorbed through GI with low bioavailability (approxi-

mately 5%) after orally taken, due to its instability in stomach acidic pH and CYP450 biotransformation in liver. In addition, doxorubicin can induce cytotoxicity in normal tissue. Currently, doxorubicin is commercially available for cancer treatment in injection dosage form. Due to its lipophilicity, doxorubicin moves through plasma membrane into the cells via passive diffusion, and its extent of tissues/cellular permeation and cellular retention can be limit by the existence of efflux transporters particularly P-gp. Apparently, doxorubicin is extensively distributed to several organs such as liver, heart, kidney after injection. Being the efflux transporters, P-gp has a significant impact on doxorubicin distribution to certain target tissues such as brain, testes [109, 110]. Certain P-gp inhibitors such as PSC-833, piperine capsaicin, resveratrol, silymarin and quercetin were reported their influence on the pharmacokinetics and tissue distribution of doxorubicin in animal models [85, 110]. Capsaicin was reported to significantly increase the extent of doxorubicin accumulation in mice brain after iv injection probably through inhibition of P-gp at blood brain barrier [110]. In addition, piperine and capsaincin,

**3.1 P-gp effects on doxorubicin's Pharmacokinetics aspect**

*DOI: http://dx.doi.org/10.5772/intechopen.95553*

later develop and lead to treatment failure.

#### *Overcoming P-Glycoprotein-Mediated Doxorubicin Resistance DOI: http://dx.doi.org/10.5772/intechopen.95553*

*Advances in Precision Medicine Oncology*

*2.2.2.2.1 MicroRNA and RNA interference (RNAi) technologies*

death in MDA-MB-231 breast cancer cells [93, 94].

mediated toxicity in this cells [97].

Application of microRNA and RNAi technologies with either small-interfering RNA (siRNA) or small hairpin RNA (shRNA) to specific silence MDR1 expression in cancer cells with MDR phenotype has been demonstrated their effectiveness in down-regulation of MDR1 and P gp expression with paralleled increases drug accumulation and improved sensitivity to treatment. MicroRNAs (miRNAs) are small non-coding RNA molecules that can inhibit ABCB1 mRNA translation processes [93, 94]. A number of miRNAs have been studied on their ability to down-regulate P-gp expression and restore cell sensitivity to P-gp drug substrates in drug resistant cells [34]. For example, miRNA-4539 could increase doxorubicin-mediated cell

The RNAi technologies involve either transient gene-silencing by siRNA or stable inhibition by MDR1 shRNA-transfected on plasmid DNA of MDR cancer cells. Treatment with siRNA against MDR1 increases drug-mediated cytotoxicity in various MDR cancer cells such as paclitaxel in MDR1 ovarian cancer cells and doxorubicin in doxorubicin-resistant breast cancer cells [95]. In addition, siRNA was able to significant reduced size of doxorubicin-resistant xenograft in a mouse model [96]. MDR1 ShRNA transfected in taxol-resistant human ovarian cancer cell line A2780 effectively down-regulated P-gp expression, and enhanced paclitaxel-

Selective suppression of P-gp/MDR1 expression with either microRNA or RNAi technologies offers the novel approach to specifically combat P-gp-based MDR in cancer, and re-sensitize the MDR cells to chemotherapeutic agents. However, for their therapeutic applications, there are several challenges required especially the effective miR/RNAi delivery to target cancer cells, design of expression vectors for

Numerous small molecules particularly those in the fourth generation of P-gp inhibitors such as curcumin, ginsenoside, quercetin and resveratrol have been demonstrated their ability to reduce P-gp function in the MDR cancer cells via down-regulation of P-gp expression [83–85]. By targeting at the signaling pathways related to transcription process of MDR1, several plant-based compounds suppress P-gp expression in the resistance cells and improve chemo-sensitivity to anticancer drugs. For instance, the P-gp modulating effect of asiatic acid, ginsenoside, isoquinoline alkaloids (e.g. cepharantine, tetrandine) resulted from their blockade of MAPK/ERK1/2 or PI3K/Akt pathways in MDR cancer cells [86, 98–101]. Another isoquinoline alkaloid berberine inhibited P-gp expression and enhanced doxorubicin-mediated toxicity in MCF-7 cells through down-regulation of AMPK-HIF1α signaling cascade [102]. Anti-MDR property of natural curcuminoids (e.g., curcumin, bisdemethoxycurcumin) involved with inhibition of human MDR1 gene expression in MDR cervical carcinoma KB-V1 cells [103]. In addition, certain compounds such as a natural marine product Et743 inhibit MDR1 transcription via

Doxorubicin is one of the most effective cytotoxic anticancer drugs. This drug has been used for combating various types of cancers such as cancers of breast, ovary, prostate, stomach, thyroid; small cell cancer of lung; squamous cell cancer of

shRNA, systemic stability and degradation, and safety of patients.

*2.2.2.2.2 Small molecules as P-gp down-regulator*

blocking its promoter activation [104].

**3. Doxorubicin and P-gp**

**196**

head and neck; multiple myeloma; Hodgkin's disease; lymphomas; acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML). Unfortunately, the uses of doxorubicin can be limited because of its dose-related toxicity (e.g., nausea, vomiting, hair loss, leucopenia, cardiomyopathy, heart failure) and high MDR incidence [105, 106]. Despite the good clinical therapeutic responses are seen in patients receiving doxorubicin in the earliest stage of treatment, multi-drug resistance may later develop and lead to treatment failure.

One of the major mechanisms responsible for doxorubicin-induced MDR is upregulation of MDR1/P-gp expression. Doxorubicin is an anthracycline derivative with a four-membered ring system containing an anthraquinone chromophore, and an aminoglycoside (**Figure 1**). This molecular structure accommodates its interaction with major MDR efflux transporters in the ABC superfamily proteins. It has been well established that doxorubicin and other anthracycline derivatives are P-gp substrates with ability to up-regulate P-gp/MDR1 expression after repeated exposure in various cancer cells such as breast and lung cancers as well as in vivo and in clinical settings [66, 107, 108]. For instance, lung perfusion with doxorubicin resulted in an increase of MDR1 RNA in patients with sarcoma pulmonary metastases [18]. The P-gp-overexpressed cancer cells would have intracellular doxorubicin concentration below its effective threshold level. Consequently, cancer cells increasingly survive from doxorubicin-mediated cytotoxity. In this circumstance, titrating dose up to overcome MDR may not enable to achieve a successful outcome due to dose-limiting toxicity. Because the adverse effects of doxorubicin and other anti-cancer drugs are mostly concentration-dependent, increasing doses can produce higher degree of severity and unendurable adverse events, leading to patient's intolerability and even fatal outcome. Addition of other cytotoxic drugs into doxorubicin-based regimens may not also enable to obtain a chemotherapeutic success, if those drugs are also substrates of the MDR transporters.

Generally, clinical efficacy of doxorubicin depends on its pharmacokinetics after systemic exposure influencing (1) the therapeutic concentration at target organs, and (2) the homogeneity of drug distribution in the cancerous tissues particularly solid tumor. In addition, it is very critical that doxorubicin accumulates within the targeted cancer cells at the level greater than its cytotoxic threshold to elicit its pharmacological actions.
