**4.1. Combined immunosuppressive therapy**

Mycophenolic acid is primarily metabolized by UDP-glucuronyl transferases (UGT1A7/8/9, UGT2B7), forming the major 7-*O*-mycophenolic glucuronide that is pharmacologically inactive and to the minor acil-glucuronide that has pharmacological activity comparable to the mycophenolic acid [52, 53]. The major proportion of the glucuronide conjugates is excreted in urine, whereas a smaller proportion that is eliminated *via* bile is metabolized by bacteria in the gut, and the deconjugated mycophenolic acid can be reabsorbed (enterohepatic circulation) [54]. Furthermore, in patients' blood and urine, a minor demethylated metabolite (6-*O*-demethylmycophenolic acid) was also detected that was proved to be produced by CYP3A enzymes.

416 Organ Donation and Transplantation - Current Status and Future Challenges

At the beginning of transplantation history, glucocorticosteroids were the primary immunosuppressive agents in the rejection prophylaxis strategy, and nowadays, they are still the first-line agents for treatment of graft rejection. The high-dose glucocorticoids given in peritransplantation are tapered to low doses in the maintenance phase, aiming the steroid-free immunosuppression regimens because of serious adverse effects of glucocorticoids developing in long-term therapy. Acute rejection is generally treated with methylprednisolone, whereas the maintenance therapy applies either methylprednisolone or prednisone. Corticosteroids activate the cytosolic glucucorticoid receptor and modulate several cellular functions, including transcription of genes involved in proliferative and inflammatory processes. The activated receptor inhibits the transcription of NF-kB and activator protein 1 dependent genes, including proinflammatory cytokines (**Figure 1**). This process leads to the depletion of T-cells and

Regioselective and stereospecific hydroxylation of corticosteroids at several positions (at carbon 2, 6, 7, 15, 16, and 21) are catalyzed by CYP3A enzymes. Additionally, dual effect of corticosteroids on CYP3A enzymes has been demonstrated: (1) corticosteroids can competitively inhibit the function of CYP3A [56], and (2) they can induce CYP3A transcription. Activated glucocorticoid receptor upregulates the expression of nuclear receptors (PXR and CAR) that are involved in transcriptional regulation of CYP3A genes. Moreover, the proximal promoter region of CYP3A4 gene contains glucocorticoid responsive element, which directly binds activated glucocorticoid receptor [18, 57]. As a consequence of increased expression and activity of CYP3A enzymes, metabolic drug interactions can be expected upon concomitant treatment

Although calcineurin inhibitor–based immunosuppression efficiently prevents rejection, adverse reactions of ciclosporin and tacrolimus, primarily nephrotoxicity, prompt the discovery of novel agents with immunosuppressive activity [58]. Two investigational agents with low molecular weight should be mentioned: voclosporin and sotrastaurin. Voclosporin, a next-generation calcineurin inhibitor, is an analogue of ciclosporin with a single carbon extension added to the amino acid-1 of ciclosporin. Voclosporin displays higher binding affinity to cyclophillin A than ciclosporin leading to more potent inhibition of calcineurin [59]. Furthermore, it has a favorable safety property that it appears to be less toxic than currently available calcineurin

with drugs that require CYP3A activity for their metabolism.

**3.6. Novel investigational immunosuppressant agents**

**3.5. Corticosteroids**

macrophage dysfunction [55].

Transplant recipients' immunosuppressive therapy is often a multidrug therapy, primarily in the early postoperative period, which constitutes a challenge for clinicians to consider the complexity of drug interactions. Due to the fact that the metabolism of immunosuppressants with low molecular weight is catalyzed by the same enzymes (CYP3A4 and CYP3A5), the blood concentrations, elimination half-lives, and consequently, the efficacy or toxicity of certain immunosuppressant agents are expected to be modified during concomitant treatment. Therefore, during multidrug therapy or during withdrawal of any of the immunosuppressive drugs, special attention is required for optimal dosing for therapeutic concentrations. Each modification in immunosuppressive regimens can lead to changes in blood concentration of a drug (**Table 2**).

Calcineurin inhibitors are often applied in combination with mTOR inhibitors. Since both mTOR inhibitors and calcineurin inhibitors are substrates of CYP3A enzymes and can inhibit CYP3A activities, reduction of calcineurin inhibitor doses is recommended. Standard doses of ciclosporin were observed to decrease the clearance of sirolimus or everolimus more substantially than the doses of tacrolimus [45]. The major drawback of calcineurin inhibitor therapy is the risk of nephrotoxicity which appears to be dose dependent. The combination of low calcineurin inhibitor doses with mTOR inhibitors was found to be beneficial regarding retaining low rejection rates and lowering the risk of nephrotoxicity [44, 63]. To avoided renal dysfunction, the complete substitution of calcineurin inhibitors for mTOR inhibitors was attempted; however, the substitution showed an increase in graft failure in patients treated with merely mTOR inhibitors [64].

Corticosteroids have been demonstrated to induce the expression of the efflux pump transporter ABCB1 (P-glycoprotein) playing a main role in intestinal drug absorption and of CYP3A enzymes responsible for the metabolism of the majority of drugs [18, 65]. Therefore, the concomitant treatment of calcineurin inhibitors or mTOR inhibitors with corticosteroids can be expected to decrease the blood concentrations of tacrolimus/ciclosporin or of sirolimus/everolimus. Although the evidence for clinically significant interactions between corticosteroids and ciclosporin or mTOR inhibitors is limited, clear clinical effect of corticosteroids on tacrolimus exposure has been demonstrated [66, 67]. This also implies that dose reduction or cessation of corticosteroids leads to an increase in blood concentrations of tacrolimus, requiring dose

**Immunosuppressant Drug interactions Consequences**

*Antifungals*:

itraconazole

*Antibiotics*:

azithromycin

*Antiviral agents:*

*Antihypertensive agents*:

*Antidiabetic agents:*

*Psychopharmacons*:

*Herbs*:

6-mercaptopurine Azathioprine

fluconazole, voriconazole,

clarithromycin, erythromycin,

ciclosporin Increased blood levels of ciclosporin and mTOR

prednisolone Decreased blood levels due to enhanced

of rejection

ketoconazole Increased blood levels of mTOR inhibitors;

derivatives

rifampicin CYP3A4 induction; enhanced metabolism of

ritonavir Irreversible inhibition of CYP3A4; increased blood

diltiazem, verapamil, amlodipine Irreversible inhibition of CYP3A4, formation of

troglitazone, rosiglitazone CYP3A4 induction; enhanced metabolism of

carbamazepine, valproic acid CYP3A4 induction; enhanced metabolism of

St John's wort CYP3A4 induction; enhanced metabolism of

allopurinol Inhibition of xantine oxidase; myelotoxicity

Mycophenolate Ciclosporin Inhibition of enterohepatic circulation, decrease in

grapefruit, pomelo Irreversible inhibition of CYP3A4; increased blood

levels of sirolimus/everolimus

blood levels of mycophenolic acid

inhibitors; increased risk of nephrotoxicity

replacement of ketoconazole to other azole

combination is contraindicated

levels of sirolimus/everolimus

levels of sirolimus/everolimus

metabolic intermediate complex;

increased blood levels of verapamil

metabolism of sirolimus/everolimus, increased risk

Metabolic Drug Interactions with Immunosuppressants http://dx.doi.org/10.5772/intechopen.74524 419

Inhibition of CYP3A4; dose reduction of sirolimus, everolimus is necessary; voriconazole – sirolimus

Irreversible inhibition of CYP3A4; increased blood

sirolimus/everolimus; increased risk of rejection

Increased blood levels of sirolimus/everolimus; verapamil-sirolimus combination is associated with

sirolimus/everolimus; increased risk of rejection

sirolimus/everolimus; increased risk of rejection

sirolimus/everolimus; increased risk of rejection

Sirolimus Everolimus

**Immunosuppressant Drug interactions Consequences**

418 Organ Donation and Transplantation - Current Status and Future Challenges

*Antifungals*:

itraconazole

*Antibiotics*:

azithromycin

*Antiviral agents*:

*Lipid-lowering agents*: fluvastatin, simvastatin,

*Antihypertensive agents*:

*Antidiabetic agents:*

*Psychopharmacons*:

*Herbs*:

atorvastatin

fluconazole, voriconazole,

clarithromycin, erythromycin,

sirolimus, everolimus Increased blood levels of ciclosporin and mTOR

risk of rejection

ketoconazole Increased blood levels of ciclosporin/tacrolimus;

derivatives

rifampicin CYP3A4 induction; enhanced metabolism of

ritonavir Irreversible inhibition of CYP3A4; increased blood

diltiazem, verapamil, amlodipine Irreversible inhibition of CYP3A4, formation of

nifedipine Reversible, competitive inhibition CYP3A4 carvedilol Inhibition of ABCB1 transporter; increase

troglitazone, rosiglitazone CYP3A4 induction; enhanced metabolism of

carbamazepine, valproic acid CYP3A4 induction; enhanced metabolism of

St John's wort CYP3A4 induction; enhanced metabolism of

grapefruit, pomelo Irreversible inhibition of CYP3A4; increased blood

levels of ciclosporin/tacrolimus

fluvoxamine Inhibition of CYP3A4; contraindicated

prednisolone Decreased blood levels due to enhanced

inhibitors; increased risk of nephrotoxicity

replacement of ketoconazole to other azole

Inhibition of CYP3A4; dose reduction of ciclosporin, tacrolimus is necessary

levels of ciclosporin/tacrolimus

levels of ciclosporin/tacrolimus

risk of myopathy and rhabdomyolysis

metabolic intermediate complex;

absorption of oral ciclosporin

Irreversible inhibition of CYP3A4; increased blood

ciclosporin, tacrolimus; increased risk of rejection

Increased statin exposure by ciclosporin; incrased

Increased blood levels of ciclosporin / tacrolimus

ciclosporin/tacrolimus; increased risk of rejection

ciclosporin/tacrolimus; increased risk of rejection

ciclosporin/tacrolimus; increased risk of rejection

metabolism of ciclosporin/tacrolimus, increased

Ciclosporin Tacrolimus

primarily in the early postoperative period. Since fungal infections are a threatening cause of morbidity and mortality, the antifungal prophylaxis is an important element of posttransplant medication. The antifungal azole-derivatives are potent (some of them are very strong) CYP3A inhibitors, predicting potential metabolic drug interactions with calcineurin inhibitors, mTOR inhibitors, or corticosteroids. The most potent CYP3A inhibitor is ketoconazole, able to increase blood concentrations (AUC) of ciclosporin (> 4-fold), tacrolimus (> 2-fold), sirolimus (11-fold), everolimus (15-fold), and methylprednisolone (> 2-fold) [73, 74]. Because of the substantial increase in blood concentrations of several immunosuppressants that can be avoided by drastic reduction of immunosuppressant doses and because of other adverse effects of ketoconazole, the concomitant medication is discouraged. Fluconazole, itraconazole, and voriconazole are alternative regimens for antifungal therapy or prophylaxis; however, all three drugs are azole derivatives and have the capability to inhibit CYP3A function, albeit at a lower extent than ketoconazole [75–77]. Although the continuous immunosuppressant monitoring is highly recommended and dose adjustment (reduction) is generally required, the antifungal treatment with fluconazole, itraconazole, or voriconazole can be safely applied except for voriconazole-sirolimus combination [78]. Because of an extreme (7-fold) increase of sirolimus blood concentrations as a consequence of concomitant use of voriconazole, this combination is contraindicated. Amphotericin B, the nonazole type antifungal agent, does not influence CYP activities; therefore, no metabolic drug interactions can be expected in concomitant treatment with immunosuppressants. However, the widespread use of amphotericin B is limited because of its toxicity profile, primarily because its nephrotoxic side-effect can

Metabolic Drug Interactions with Immunosuppressants http://dx.doi.org/10.5772/intechopen.74524 421

Organ transplant patients are at high risk for developing bacterial infections that occur in 20–40% of transplants. Potential sources of infection are from hospital and community exposures, as well as from endogenous flora of patients. Among the antibiotics used for treatment of infections, the macrolide erythromycin and clarithromycin have been reported to interact with immunosuppressive agents. These macrolides are CYP3A substrates and bind to CYP3A4 enzymes, leading to a complex formation that completely inactivates CYP3A4 enzyme [79–82]. The *in vitro* findings were confirmed by clinical observations that blood concentrations of ciclosporin/tacrolimus or sirolimus/everolimus increased as a consequences of concomitant treatment with erythromycin or clarithromycin [73, 83–86]. Page et al. [87] and Mori et al. [88] have reported some potential of azithromycin for drug interaction with ciclosporin and tacrolimus; however, *in vitro* experiments demonstrated that azithromycin poorly interfere with CYP3A4 [89]. When concomitant therapy with these macrolides is necessary, blood concentrations of calcineurin inhibitors or mTOR inhibitors should be carefully monitored, and the immunosuppressant doses should be adjusted. In contrast, the macrolide rifampicin is a potent CYP3A4 inducer and can activate PXR, resulting in a substantial increase in CYP3A4 expression [90]. The increased CYP3A4 activity consequently enhances the metabolism and elimination of calcineurin inhibitors, mTOR inhibitors, and corticosteroids [91–93]. However, blood concentration–guided dose-adjustment of immunosuppressants should be applied carefully because increased metabolism can evoke elevation of toxic metabolite formation (e.g., ciclosporin).

A significant cause of graft failure still remains viral infections, which are acquired as new infection or reactivation of latent viruses. After transplantation, cytomegalovirus (CMV) is the

contribute to the renal injury by ciclosporin or tacrolimus.

**Table 2.** Clinically relevant pharmacokinetic drug interactions with immunosuppressants.

adjustment [68]. Interestingly, CYP3A5 nonexpressers with *CYP3A5\*3/\*3* genotype are more susceptible to glucocorticoid induction than *CYP3A5\*1* carriers [69]; thus, more pronounced increase in tacrolimus exposure can be expected in CYP3A5 nonexpressers after glucocorticoid withdrawal.

Clinically significant interaction between mycophenolic acid, the active metabolite of mycophenolate mofetil, and ciclosporin has been reported [70]. The mycophenolate-glucuronide metabolite eliminated into bile undergoes enterohepatic cycling because of intestinal bacterial metabolism and reabsorption of mycophenolic acid. The enterohepatic circulation, contributing to overall pharmacokinetics of mycophenolic acid by 37% in human, is inhibited by concomitant administration of ciclosporin but does not interfere with tacrolimus or sirolimus [71, 72]. In ciclosporin-mycophenolate combination therapy, the reduced blood concentration of mycophenolic acid is necessary to ameliorate by increasing dose of mycophenolate mofetil. Furthermore, special attention on optimal dosing is required during switching ciclosporinmycophenolate to tacrolimus-mycophenolate therapy and *vice versa*.


**8. Clinical impact of angiogenic inhibitors**

In contrast, DLL4 expressed in ECs stimulates Notch 3 receptors located on adjacent cells (e.g., colorectal cancer or T-cell acute lymphoblastic leukemia cells) to activate tumor progression from dormant to active phase [60]. Such findings consider Notch pathways a potential therapeutic target for the design and development of novel anti-angiogenic compounds, although the Notch cell signaling shows a mixed behavior of tumor progression and inhibition in clini-

**Figure 2.** VEGF-induced intracellular signaling in tumor angiogenesis and angiogenic inhibitors with targeted active sites: The binding of vascular endothelial growth factors (VEGF) to respective transmembrane receptors stimulates a plethora of intracellular signaling pathways which regulate nuclear transcription factors for altered gene expressions of normal cell responses including loss of tumor suppression, activation of hypoxia inducible factor (HIF-α), increased receptor tyrosine kinase activity, increased tumor cell growth, and repression of oncogene mutations. Angiogenic inhibitors to their targeted active sites are also shown with numerical circles in the figure. Only anti-angiogenic compounds approved by the US Food and Drug Administration (FDA) for the treatment of numerous solid tumors and carcinomas are depicted where circle 1 represents growth factor inhibitors (bevacizumab, aflibercept); circle 2, growth factor receptor inhibitors (sunitinib, sorafenib); circle 3, RAS inhibitors (tipifarnib, lonafarnib); circle 4, RAF inhibitors (sorafenib); circle 5, HIF-1α inhibitors (geldanamycin, chetomin, echinomycin, 2ME2); circle 6, PI3K inhibitors (wortmannin, LY294002); circle 7, AKT inhibitors (FARA-A); and circle 8, mTOR inhibitors (rapamycin and analogues). JNK = JUN N-terminal kinase; MAPK = mitogen-activated protein serine/threonine kinase, MAPKK = MAPK kinase, PDK1 = phosphoinositidedependent protein kinase-1; PLC = phospholipase C; PtdInP2 = phosphatidylinositol 4,5-bisphosphate, Ins (1,4,5) =

310 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

Transforming growth factor is a ubiquitously expressed paracrine polypeptide of approximately 25 kDa molecular weight [61]. TGF 1 to TGF 3 are three highly homologous isoforms of the polypeptide and discovered in humans and mammals [62]. TGF-β is initially synthesized as a zymogen, and after secretion, an associated peptide is proteolytically sliced to release

Active TGF-β binds to constitutively active serine/threonine kinase TGFBR2 receptors to activate TGFBR1 in a heterodimer complex which controls transcription via activation of canonical signal pathways mediated by a family of SMAD proteins (SMAD1-5) (**Figure 3b**) [64]. The

cal assays [58, 59].

inositol 1,4,5-triphosphates.

**7.3. Transforming growth factor-β (TGF-β)**

active form of the growth factor [63].

The discovery and development of angiogenic inhibitors have raised the hopes to treat a lot of tumors and carcinomas and ultimately to reduce the morbidity and mortality related to tumors and cancers [67]. Five classes of anti-angiogenic compounds have established and are still under investigation on the basis of potential antitumor drug targeted sites including proteases inhibitors (MMP synthesis inhibitors), ECs proliferation and migration inhibitors, vascular endothelial growth factor inhibitors, cell matrix protein inhibitors, and angiogenic inhibitors with unique mechanisms (**Figure 2**) [68, 69].

Although the anti-angiogenic compounds approved by the FDA show therapeutic efficacy in some categories of cancer as monotherapy, however, sufficient published data recently reveal this fact that angiogenic inhibitors are best therapeutic choices for tumors when used in combination with traditional chemotherapies [70, 71]. However, one would not expect in the first instance that angiogenic inhibitors might reduce the intratumoral delivery of cytotoxic agents (traditional chemotherapy) by decreasing perfused blood vessels with impaired blood flow and decrease drug transport in treated tumor cells [48, 72]. It would also increase tissue hypoxia and inhibit tumor cell proliferation although proliferating cells are an easy target for chemotherapy [48, 72].

To overcome such hurdles and to enhance synergistic therapeutic potential of chemo and anti-angiogenic drugs when used in combination, Kerbel proposed three mechanistic approaches in this scenario to be adopted; first, normalization of tumor microvessels by anti-angiogenic compounds [73, 74]; second, maximum tolerated dose chemotherapy during the break periods of successive courses [72, 75], and third, use of known chemotherapeutic agents having anti-angiogenic effects [72]. The additional advantages of chemotherapy while improving their anti-angiogenic effects may be grabbed by adopting "metronomic chemotherapy" which states that "the administration of chemotherapeutic agents at relatively low, minimally toxic doses on a frequent schedule of administration at regular close intervals, with no prolonged drug-free breaks [76, 77]." By such approaches, endothelial cells are directly killed, and progenitor ECs are suppressed in circulation. Furthermore, minimal use of toxic doses lowers the frequency of adverse events in treated patients [72, 76, 77]. Such treatment strategies may be adopted for a prolonged period of time with angiogenic inhibitors in the treatment of advanced solid tumors with little side effects as validated by phase II clinical trials; however, phase III clinical studies are extensively demanded in this direction [70, 71].

## **9. Conclusions**

In vivo, in vitro, and in ova assays for angiogenesis assessment are the reliable approaches in basic research and to some extent in real-world clinical practices. However, in vivo systems are difficult to perform and time consumable, and the process of quantification is much complicated than in vitro assays. Conversely, these are relatively better due to complex nature of the vascular response to the test compound. In vitro angiogenesis assays may perform in a short period and provide the accurate and reliable outcome of angiogenic processes. Mouse models based angiogenesis assays have also standardized to an improved understanding of tumor angiogenesis and lymphangiogenesis. Similarly, such models are also used to assess vasculogenesis and arteriogenesis in ischemic heart diseases, blindness, psoriasis, and arthritis. Angiogenesis assessment always plays a focal role to determine the pathogenesis and progression of certain challenging diseases in human populations in particular human cancer. An ample understanding of angiogenesis research in tumor progression, by knowing the molecular mechanisms and cellular pathways, also opens the ways to design and develop effective anti-angiogenic inhibitors. The manipulation of the human genome in a precise and predictable manner due to recently developed molecular techniques has opened new gates for the generation of more reliable models for angiogenesis studies and the testing of new therapeutic strategies.