Drug conjugates—an emerging approach to treat breast cancer

Abstract Breast cancer treatment using a single drug is associated with a high failure rate due, in part, to the heterogeneity of drug response within individuals, nonspecific target action, drug toxicity, and/or development of resistance. Use of dual‐drug therapies, including drug conjugates, may help overcome some of these roadblocks by more selective targeting of the cancer cell and by acting at multiple drug targets rather than one. Drug‐conjugate approaches include linking drugs to antibodies (antibody‐drug conjugates), radionuclides (radioimmunoconjugates), nanoparticles (nanoparticle‐drug conjugates), or to other drugs (drug‐drug conjugates). Although all of these conjugates might be designed as effective treatments against breast cancer, the focus of this review will be on drug‐drug conjugates because of the increase in versatility of these types of drugs with respect to mode of action at the level of the cancer cell either by creating a novel pharmacophore or by increasing the potency and/or efficacy of the drugs’ effects at their respective molecular targets. The development, synthesis, and pharmacological characteristics of drug‐drug conjugates will be discussed in the context of breast cancer with the hope of enhancing drug efficacy and reducing toxicities to improve patient quality of life.

increased risk of developing breast cancer. 36 Mutation(s) of a specific signaling protein may also lead to drug resistance. For example, estrogen receptor-positive breast cancer cells (MCF-7) are known to display drug resistance by modulation of the antiapoptotic protein, Bcl2, in the presence of estrogenic stimulation. 108 Resistance may also arise through activation of drug transporters that prevent the accumulation of the anticancer drug within the cell. 46 For example, anthracycline drugs are readily effluxed from cancer cells overexpressing P-glycoprotein or multidrug resistance protein. 27 In addition, cancer phenotypes in different human populations vary, and a response to a single drug may not be equally efficacious across all individuals. 84 Drug conjugates represent a growing class of anticancer agents designed to (1) increase target selectivity and reduce the bystander effect (eg, drug-antibody conjugates; Figure 2A; Table 1), (2) enhance cytotoxicity and tumor targeting (eg, radionuclide-drug conjugates, nanoparticle-drug conjugates; Figure 2B; Tables 2 and 3) or (3) enhance the potency and/or efficacy of anticancer therapy (eg, drug-drug conjugates; Figure 2C; Table 4). To overcome low efficacy, drug resistance, and/or toxicity associated with single drug use or monotherapy, dual-drug, and even triple-drug therapies are being developed or are already in clinical use. 1 For example, paclitaxel and trastuzumab combination drugs, or capecitabine and docetaxel combination drugs have been shown to reduce mortality and improve safety compared to their administration as single drugs. 78 However, most of these therapies deliver drug combinations as separate entities.
Drug conjugates offer an alternative approach in situations where a single drug or monotherapy fails. A drug-drug conjugate comprises 2 or more drugs connected by a chemical linker (Figure 2C and D). These novel types of drugs may be useful for targeted drug delivery and modulation of pharmacokinetic parameters, rendering the drugs more effective and less toxic than in isolation.
Furthermore, conjugation of 2 drugs (eg, melatonin-tamoxifen) with a noncleavable linker may produce a novel molecule with unique pharmacological characteristics, 116 (US Patent No. 08785501). Although antibody-drug conjugates comprise a large segment of conjugated drugs (see Table 1), this class of drugs will not be discussed here, and the reader is referred to recent excellent papers. 51,89 The focus of this review will be on the emerging field of drug-drug conjugates reported in the literature, their design and their pharmacological characteristics with respect to drug targets (receptors, intracellular signaling proteins) and efficacy as antibreast cancer agents.

| DRUG CONJUGATES/ HYBRID LIGAN DS
Ideally, a drug-drug conjugate should be stable in the systemic circulation before arriving at the target site, which is not only F I G U R E 1 Number of new drugs approved by FDA from 2011 to 2018 (data collected from https://www.fda.gov/Drugs/Development ApprovalProcess/DrugInnovation/default.htm) valid for drug conjugates with a noncleavable linker, but also for those formed by a cleavable spacer such that drug release will occur only at the intended site of action. 26 A noncleavable linker may also be designed to maintain its chemical integrity, allowing it to act as a single moiety/pharmacophore at its receptor or receptors. 72 Unlike combination drug therapy, in which 2 individual unlinked drugs are administered simultaneously, conjugated drug-drug therapies may offer distinct advantages therapeutically, aesthetically, or both. 63 With respect to esthetics, it may be more convenient for a patient to take a single drug conjugate instead of 2 drugs, thus improving compliance. 63 Concerning therapeutic advantages, conjugated drug therapies may improve the therapeutic index by lowering the minimum effective dose and by increasing the maximum tolerated dose compared to traditional combination therapies. 73 Drug-drug conjugates may also prevent drug resistance, as tumors are less likely to become resistant to drugs with distinct targets (ie, receptors, and/or intracellular signaling proteins). 1,16 A drug conjugate may display superior activity (potency, and/or efficacy) compared to a single drug; this may be a result of improved bioavailability 119 or through additive or synergistic action of the 2 drugs working in concert. 97 Moreover, 2 conjugated pharmacophores targeted for 2 different receptors may show higher affinity by coactivation in the same cells. 48 For example, paclitaxel and doxorubicin drug conjugates showed superior efficacy and safety compared to the combination by acting synergistically because it delivers both drugs to the same target site at a synergistic ratio. 71 The increase in potency of a drugdrug conjugate may also produce less toxicity as the recommended dose could be lowered. 5,24,61,62 For example, chimeric c-Src kinase and histone deacetylase inhibitors 59 and the multiacting EGFR/HER2 and histone deacetylase inhibitor CUDC- 101 12 were reported to be not only more active than the combination of the 2 constituting parent compounds, but also less toxic with a higher improved therapeutic index. These findings indicate that molecular hybridization does not necessarily lead to the additive toxicity often observed for the combination therapy.

| Drug targets
Stability and efficacy of drug conjugates need to be carefully considered during their design. Concerning stability, conjugates containing a cleavable linker should be designed to release drugs only at the target site. 24 With respect to improved efficacy, conjugates can be designed to target breast tumors with unique phenotypes, such as luminal A (ER+/HER2+; PR+/HER2−), luminal B (ER+/HER2+; PR+/ HER2+), HER2 (ER-/PR-/HER2+), or to target specific intracellular signaling proteins in triple-negative breast cancer, (eg, mTOR, PI3K, PARP, TROP-2, VEGFR, EGFR, FGFR, CYP17A1, MEK, AKT, anti-CD27, anti-CD52, hedgehog). 124 In such cases, combining 2 drugs with different pharmacological targets ensures their equivalent F I G U R E 2 Possibilities of drug conjugates. A = antibody targets-specific receptor (mentioned in table 1), B = radioactive isotope (mentioned in Table 2), C = chelating agent (DOTA, EDTA) D = cytotoxic drug (mentioned in Table 1), E = binds with a receptor (tamoxifen, endoxifen), F = regulates a signal pathway (anti-NF-КB, DNA intercalator), G = regulates an enzyme (kinase inhibitors, HDAC inhibitors), I = endogenous compound (melatonin), J = nanoparticle (gold). L = linker for conjugation (valine-citrulline, hydrazine) Antibody-cytotoxic drug conjugates (ADCs) are the most widely investigated drug conjugates to treat breast cancer. 89 In general, a cytotoxic drug is attached to a monoclonal antibody that is specific for the target receptor ( Figure 2A). The antibody binds to the receptor of the cancer cell where the cytotoxic drug is intended to exert its actions. Therefore, the cancer cells should ideally densely express the receptor for antibody binding. Although over 55 ADCs are currently in clinical trials, 13 only 3 ADCs have been approved by the FDA. 77 However, gemtuzumab ozogamicin (marketed as Mylotarg ® by Wyeth-Ayerst) was withdrawn in 2010 due to increased patient mortality and demonstrating no clinical benefit over conventional therapy, which leaves only 2 ADCs available for clinical use. One of them is for HER2-positive metastatic breast cancer-the trastuzumab-emtansine conjugate marketed as Kadcyla ® by Genentech and Roche. Another one is brentuximab-vedotin (Adcetris ® marketed by Seattle Genetics) for Hodgkin lymphoma or anaplastic large cell lymphoma. In many ways, ADCs may exert potential benefits over conventional treatment. For example, highly cytotoxic drugs might become safer for normal cells when they are bound to cancer cell-specific antibodies. 115 delivery to the cancer tissue, which may not be the case if the 2 combination drugs differ in pharmacokinetic properties of tissue distribution and elimination.

| Linker design
The following questions need to be addressed when designing chem- However, it is also possible that the conjugate will exert its action by maintaining the integrity of the linker until it reaches its target site(s) where it will then be broken down chemically or enzymatically depending on the structure of the linker; this type of linker is referred to as a cleavable linker. 74 One drawback to the type of drug conjugate is that the local release of drugs at their target site(s) may also impact neighboring "nontarget" sites, which may cause toxicity depending upon the cellular milieu. 47 Drugs conjugated with a chemically cleavable linker are released under specific biochemical environments. A hydrazone linker is usually stable in standard blood pH conditions (pH 7.4) and hydrolyzed under acidic conditions, such as in lysosomes or in the more acidic tumor microenvironment. 30 For example, adriamycin conjugated to PEO-b-PAsp copolymers with a hydrazone linker exhibited release of the active drug at pH levels below 5. 44 However, one drawback to this type of linker may be its instability in acidic buffers and excipients as observed for hydrazone linkers. 52 In a recent study, a drugantibody conjugate using this type of linker [ie, gemtuzumab ozogamicin (Mylotarg ® )] was withdrawn due to its narrow therapeutic index, poor plasma stability, and lack of benefit over conventional chemotherapy. 30,74,88 Even though this was with a drug-antibody conjugate, these findings need to be taken into consideration when designing a drug-drug conjugate.
Chemical linkers containing disulfide bonds are also chemically cleavable. For example, disulfide bonds may be reduced in vivo by glutathione. As glutathione levels are relatively low in plasma (2-20 μmol/L) compared to the cytoplasm (0.5-10 mmol/L) 75,115,118 disulfide bond-containing linkers remain relatively stable in blood.
This property improves the pharmacokinetic profiles of the conjugates compared to their unconjugated antibody component. For Radiation emitted from a radionuclide can be used to kill cells. Radioactive compounds can also attack noncancerous cells; therefore, targeted delivery of the radionuclide with the help of a monoclonal antibody is desirable. The radionuclide can be linked to a monoclonal antibody by a linker, or the antibody could be labeled with radioisotope by a chelation method ( Figure 2B).
example, the disulfide linker containing a doxorubicin-gold drug conjugate exhibited greater intracellular uptake than free doxorubicin in multidrug-resistant cancer cells. 41 One point to consider when creating drug conjugates with a disulfide linker is its stability during the sterilization process. Heat generated during the sterilization process can break down disulfide bonds. 68 This obstacle could be overcome through filter sterilization. 31 Linkers may be designed to be degraded by enzymes within the breast cancer cell to control drug delivery at the target site; this may avoid premature release into the systemic circulation due to factors such as low pH. 30  The uptake of nanoparticles by a tissue depends on the hydrophobicity of that nanoparticle. For example, nanoparticles deposited in certain organs such as the liver, spleen, and reticuloendothelial system correlate positively with the increasing hydrophobicity of the polymer. 37 Although several nanoparticle-based drug delivery systems have been developed, only albumin-bound paclitaxel nanoparticle (Abraxane ® ) was approved by FDA for metastatic breast cancer and nonsmall cell lung carcinoma. 69 Nanotechnology can also be effectively used in breast cancer treatment. Nanoparticle conjugates may show increased potency by penetrating the cells by endocytosis instead of the diffusion method used for a single drug. 28 This could be a mechanism to avoid efflux by drug transporters such as P-glycoprotein. 14 In a phase III clinical trial, paclitaxel nanoparticles bonded with albumin showed superior efficacy and safety compared to paclitaxel dissolved in castor oil. 39   The triple drug conjugate platinum-acridine-endoxifen exhibited higher cytotoxicity compared to a tamoxifen/cisplatin combination therapy, which warrants further investigation. 23 Table 4).  (Table 4). 56,58 An estrogen receptor antagonist, oxabicycloheptene sulfonate,  and panabinostat. 70 The concept of drug conjugates can also be used in the development of targeted drug delivery systems. Targeted drug delivery systems are generally stable in the systemic circulation, and they might be designed to release the cytotoxic drugs only after internalization into cancer cells. 49 Some drug conjugates were specifically designed to deliver the cytotoxic drug at the target site using a pharmacologically inactive drug. Examples of tamoxifen drug conjugates targeted for specific drug delivery are described in Table 3.

| CONCLUSION
Hundreds of receptors and signaling pathways are dysregulated in breast cancer, a highly heterogeneous disease that exploits multiple targets, such as receptors from different classes and myriad signal transduction cascades. 42 Because of these factors, breast cancer drugs need to be selective and personalized based on the patient's genotype or phenotype. Drug-drug conjugates offer a partial solution to these issues because conceptually they target multiple proteins and signaling pathways at the same time within a cancer cell.  Tables 1-4, several in vitro and preclinical examples suggest that conjugates may harbor a therapeutic advantage over a single drug or standard drug combination treatment regimens.
As almost 60% of breast cancer patients are hormone receptorpositive, antiestrogen drug conjugates hold promise as effective therapeutics. 32 Although some tamoxifen drug conjugates display higher potency compared to tamoxifen alone, there remains the risk of tamoxifen drug resistance. Therefore, testing these conjugates in tamoxifen-