A Novel Paclitaxel Conjugate with Higher Efficiency and Lower Toxicity

International Journal of Molecular Sciences

Article A Novel Paclitaxel Conjugate with Higher Eﬃciency and Lower Toxicity: A New Drug Candidate for Cancer Treatment

1,2, 3,4, 3,5,6, Mizied Falah †, Mahmoud Rayan † and Anwar Rayan * 1 Institute for Medical Research, Galilee Medical Center, Nahariya 2210001, Israel; [email protected] 2 Faculty of Medicine in the Galilee, Bar-Ilan University, Safed 1311502, Israel 3 Drug Discovery Informatics Lab, QRC—Qasemi Research Center, Al-Qasemi Academic College, Baka El-Garbiah 30100, Israel; [email protected] 4 HydroMap Limited Company, Kabul 2496300, Israel 5 Institute of Applied Research, Galilee Society, Shefa-Amr 20200, Israel 6 IDD Therapeutics, Wadi El-Haj 13, P.O. Box 1252, Nazareth 17111, Israel * Correspondence: [email protected] These authors contributed equally to this work. † Received: 29 August 2019; Accepted: 5 October 2019; Published: 8 October 2019

Abstract: Paclitaxel-lipoate (IDD-1040) is a conjugate formed by the chemical joining of the two compounds, by condensing a lipoic acid moiety to the C20 of paclitaxel. IDD-1040 was evaluated for its anti-tumor activity and potential druggability, using an in vivo non-small-cell, lung cancer (NSCLC) xenograft mouse model. In the in vivo studies, IDD-1040 showed a maximum tolerated dose (MTD) of 250 mg/kg compared to paclitaxel (PTX), with an MTD of 20 mg/kg. Most interesting, IDD-1040 demonstrated higher anti-tumor activity, and its inhibitory activity on tumor volume (cell growth) was dose-dependent. That anti-tumor activity persisted for two weeks after cessation of IDD-1040 treatment, as opposed to PTX cessation, after which the tumor relapsed, conﬁrming that IDD-1040 exhibits superior tumor inhibition. Similar to PTX treatment, no marked body weight decrease was observed during IDD-1040 treatment, indicating a low toxicity proﬁle. The increase in animal body weight noted over time was due to the increasing weight of tumors, recorded in all the mouse test groups. The results also showed that mortality rate of mice was reduced by treatment with IDD-1040, more so than with PTX. Furthermore, in a preliminary study on the ex vivo distribution of IDD-1040, neutropenia was primarily concentrated in the liver 1 h after injection, and most of the drug was metabolized by the liver in 24 h. All of these results demonstrate IDD-1040’s great potential as a candidate drug for cancer treatment.

Keywords: paclitaxel; lipoic acid; paclitaxel-lipoate conjugate; IDD-1040; anticancer drug; anti-tumor activity

1. Introduction Paclitaxel (PTX), known by its brand name as Taxol®, is a diterpenoid pseudoalkaloid that consists of a taxane ring and an N-benzoylphenylisoserine group. It was first isolated from the tree bark of the Pacific yew, Taxus brevifolia, and continues to be largely used as an antitumor drug [1]. PTX’s characteristic structure is somewhat different from those of the other taxanes, due to a complex C-13 side chain attached to the taxane ring, which is important for its anti-tumor activity (Figure1, left side). Preclinical studies have shown many promising results against many tumors, and due to its increasing demand, PTX is currently considered one of the most important anti-tumor drugs in clinical use. It

Int. J. Mol. Sci. 2019, 20, 4965; doi:10.3390/ijms20194965 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 4965 2 of 15 isInt. used J. Mol. to Sci. treat 2019 non-small, 20, x FOR PEER cell REVIEW lung cancer [2], cervical cancer [3], brain cancer [4], ovarian cancer3 of [ 515], breast cancer [6], and other cancers.

Figure 1. The chemical structure of paclitaxel (left side), and IDD-1040 (right side). Figure 1. The chemical structure of paclitaxel (left side), and IDD-1040 (right side). The mechanism by which PTX exerts its anti-tumor activity is stabilization of the dynamic 2. Results and Discussion cytoskeletal structures of microtubules during the mitotic phase of cell division. In response to PTX, tumorThe cells, synthesis which yield have of an IDD accelerated-1040 product, cell division as explained rate, are in the unable method to forms section, the highly was 69%. ordered The arrangementpurity of the ofproduct, microtubule as assessed arrays, by characteristic HPLC, was slight of eukaryoticly above cells97%. duringFigure cell2 depicts division, the sotypical that theHPLC dynamic chromatogram spindle assemblyobtained ofusing microtubules the isocratic is damaged,mobile phase resulting (methanol:water in cell death—82:18, [7–9]. v PTX,/v) at ina addition,flow rate blocksof 1.0 mL/min several otherwith UV microtubule-based detection at 225 processes nm of a 5within μL injection. the cytoplasm A blank that sample are fundamentalis shown on tothe the left development and a sample and receiving maintenance 200 μg/mL of cells IDD [10-1040]. It on also the a ffright.ects major cell components involved in the processes of cell shaping and the transporting of vesicles, mitochondria, and other intracellular proteins, and it interferes with the signaling pathways that regulate processes of cell growth [9,11,12]. PTX also affects microtubule-associated proteins (MAPs) that govern the highest level of microtubule dynamics, through which chromosomes are correctly fixed, separated, and segregated during the mitotic phase of cell division [13]. Upon binding, suppressing microtubules dynamics, and preventing the progression of mitosis, PTX causes the arrest of tumor cells in the G2/M phase of the cell cycle, and thus halts the division and proliferation of these cells. Not only does PTX affect microtubules and interfere with the cell cycle progression, but it also causes changes in signaling related to apoptosis, so that cell death can occur independently of microtubule stabilization and mitotic inhibition. Moreover, it has been found that PTX is capable of binding a protein that regulates apoptosis and induces the phosphorylation of proteins—resulting in cell death [14]. Clinically, the efficacy and safety profiles of the taxanes in general, and PTX in particular, have been well established to certain extents in cancer treatment. Although PTX is still used in clinics to treat a variety of tumors [15–19], its use is associated with serious reactive oxygen species generation [20] and other common side effects, such as neutropenia—the most important reversible hematologic toxicity, which is dose and schedule-dependent, and neuropathy that often produces irreversible toxicity,Figure aff ecting2. HPLC activities chromatograms of daily of living a blank and sample overall (left) quality and a sample of life [receiving17]. Neuropathic 200 μg/mL symptoms IDD-1040 are generally(right). managed by delaying doses or reducing or discontinuing PTX administration, causing a negative impact on tumor treatment results [21]. Taxane-induced neuropathy, which develops through 2.1. NMR Assignments the inhibition of microtubule function within neurons, has been clinically studied using the drug with various modifications or with other agents or vitamin combinations [17,22]. None of these interventions The numbers of atoms are shown in figure 3. has demonstrated a significant improvement in neuropathic symptoms [17]. In1H a-NMR recent (CDCl study,3 the): 1.19 oral (s, administration CH3, H-16), 1.25 of lipoic (m, 2H, acid H in-5’ patients), 1.28 (s, with CH painful3, H-17), diabetic 1.28 (m, neuropathy 2H, H-6’), has1.65 been (m, 2H, found H- to5’), have 1.64 a ( clinicallym, 4H, H4 significant’ and H6’), impact 1.71 (s. on CH the3, controlH-18), of1.97 neuropathy (m, 2H, H9 and’), 1.99 quality (ddd of, life1H, [23 H].- As6-), a strong,1.97 (s, naturalCH3, H- antioxidant19), 2.25 (s, showing3H, OAc- extensive10), 2.36 (m, biological 2H, H-4 activity,’), 2.40 ( lipoicdd, 1H, acid H- (LA)14), has2.44 also (dd, been 1H, H-14), 2.48 (s, 3H, OAc-4), 2. 45 (m, 3H, H8’and H-10’), 2.56 (ddd, H6-), 3.81 (d, 1H, H-3), 4.15 (d, 1H, H-2O), 4.22 (d, 1H, H-2O), 4.46 (ddd, 1H, H-7), 5.02 (d, 1H, H-2’), 5.31 (s, 3H, H-10), 5.58 (d, 1H, H-2), 6.24 (t, 1H), 6.33 (s, 3H, H-10), 6.90 (d, 1H, NH-3’), 7.36–7.44 (tt, 1H, p-PH-2, 5H), 7.42 (m,

Int. J. Mol. Sci. 2019, 20, 4965 3 of 15 used in combination with other drugs to treat tumors and minimize the danger of neuropathy [24,25]. LAInt. J. is Mol. a naturalSci. 2019, compound20, x FOR PEER widely REVIEW present in the body, and due to its chemical properties,3 it of has 15 high activity in both aqueous and lipid media. It acts both in the extracellular space and in the intracellular regions of cells, hence it has a wide range of pharmacological applications [26,27]. It is considered one of the most effective antioxidants used in daily clinical practice [28], as it exhibits protective effects against ischemia-reperfusion injuries in various organs, following the production of reactive oxygen species (ROS), which otherwise causes tissue damage [29,30]. Moreover, LA plays a protective role in diabetes and has been found to be essential in the treatment of diabetic neuropathy and cataracts [31,32]. Not only is LA effective in many trials, but it is also safe upon administration. No upper limit of LA administration has been defined in clinical use, and no adverse effects have been noticed over time at higher doses [33,34]. In regard to cancer treatment, recent studies, among many others, have confirmed that LA can inhibit cell proliferation in cervical, breast, colon, prostate, and lung cancers [24,35–38]. Most importantly, the combination of LA and PTX has shown an enhanced inhibitory effect on breast cancer cell proliferation [25]. Showing great promise, we think that this may provide a basis for qualifying LA as a chemotherapeutic drug of choice. This study aimed to explore the possible consideration of IDD-1040 as an anticancer drug candidate. IDD-1040 (Figure1, right side) was chemically synthesized by condensing an LA moiety to paclitaxel, forming a new paclitaxel ester, and the compound was studied for its inhibitory effect on tumor cells in vitro and in vivo (using xenograft modelsFigure with 1. The intravenous chemical structure injection). of paclitaxel (left side), and IDD-1040 (right side).

2. Results and DiscussionDiscussion The synthesissynthesis yieldyield ofof IDD-1040IDD-1040 product,product, asas explainedexplained inin thethe methodsmethods section,section, waswas 69%.69%. TheThe puritypurity ofof the the product, product, as as assessed assessed by byHPLC, HPLC, was was slightly slight abovely above 97%. 97%. Figure Figure2 depicts 2 depicts the typical the HPLCtypical chromatogramHPLC chromatogram obtained obtained using the using isocratic the mobileisocratic phase mobile (methanol:water—82:18, phase (methanol:waterv/v—) at82:18, a ﬂow v/v rate) at ofa 1.0flow mL rate/min of with1.0 mL/min UV detection with UV at225 detection nm of aat 5 225µL injection.nm of a 5 AμL blank injection. sample A blank is shown sample on the is shown left and on a samplethe left receivingand a sample 200 µreceivingg/mL IDD-1040 200 μg/mL on the IDD right.-1040 on the right.

Figure 2. HPLC chromatograms of a blank sample (left) and a sample receiving 200 µg/mL Figure 2. HPLC chromatograms of a blank sample (left) and a sample receiving 200 μg/mL IDD-1040 IDD-1040 (right). (right). 2.1. NMR Assignments 2.1. NMR Assignments The numbers of atoms are shown in Figure3. 1 The numbersH-NMR of (CDCl atoms3): are1.19 shown (s, CH in3 ,figure H-16), 3 1.25. (m, 2H, H-5’), 1.28 (s, CH3, H-17), 1.28 (m, 2H, H-6’), 1.65 (m, 2H, H-5’), 1.64 (m, 4H, H4’ and H6’), 1.71 (s. CH3, H-18), 1.97 (m, 2H, H9’), 1.99 (ddd, 1H, 1H-NMR (CDCl3): 1.19 (s, CH3, H-16), 1.25 (m, 2H, H-5’), 1.28 (s, CH3, H-17), 1.28 (m, 2H, H-6’), H-6-β), 1.97 (s, CH3, H-19), 2.25 (s, 3H, OAc-10), 2.36 (m, 2H, H-4’), 2.40 (dd, 1H, H-14β), 2.44 (dd, 1H, 1.65 (m, 2H, H-5’), 1.64 (m, 4H, H4’ and H6’), 1.71 (s. CH3, H-18), 1.97 (m, 2H, H9’), 1.99 (ddd, 1H, H- H-14α), 2.48 (s, 3H, OAc-4), 2. 45 (m, 3H, H8’and H-10’), 2.56 (ddd, H6-α), 3.81 (d, 1H, H-3), 4.15 (d, 6-), 1.97 (s, CH3, H-19), 2.25 (s, 3H, OAc-10), 2.36 (m, 2H, H-4’), 2.40 (dd, 1H, H-14), 2.44 (dd, 1H, H-14), 2.48 (s, 3H, OAc-4), 2. 45 (m, 3H, H8’and H-10’), 2.56 (ddd, H6-), 3.81 (d, 1H, H-3), 4.15 (d, 1H, H-2O), 4.22 (d, 1H, H-2O), 4.46 (ddd, 1H, H-7), 5.02 (d, 1H, H-2’), 5.31 (s, 3H, H-10), 5.58 (d, 1H, H-2), 6.24 (t, 1H), 6.33 (s, 3H, H-10), 6.90 (d, 1H, NH-3’), 7.36–7.44 (tt, 1H, p-PH-2, 5H), 7.42 (m,

Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 4 of 15

5H, m-Ph3, mPh2,p-Ph2), 7.55 (m, m-Ph-3, m-Ph1, o-Ph2, 4H), 7.62 (tt, 1H, p-Ph1), 7.77 (dd, 2H, o- Ph3), 8.15 (dd, 2H, o-Ph1).

Int.2.2. J.13 Mol.C-NMR Sci. 2019 (CDCl, 20, 49653) 4 of 15 203.5 (C-9), 172.2 (C-1), 171.1 (C=O, OAc-10), 169.9 (C=O,OAc-4), 168.1 (C=O, lipoate), 167.0 1H,(C=O, H-2O Ph3),β), 167.01 4.22 (d, ( 1H,C=O, H-2O Ph1),β), 142.8 4.46 (ddd,(C-12), 1H, 136.9 H-7), (q 5.02-Ph3), (d, 133.8 1H, H-2’), (p-Ph1),133.1 5.31 (s, 3H, (C11), H-10), 132.7 5.58 ( (d,qPh2), 1H, H-2),130.01 6.24 (o-Ph1), (t, 1H), 129.1, 6.33 128.7, (s, 3H, 127, H-10), 127.7, 6.90 126.5 (d, ( 1H,Aromatic NH-3’), C), 7.36–7.44 83.2 (C-4), (tt, 81.1 1H, ( p-PH-2,C-1), 79.1 5H), (C- 7.4220), 76.2 (m, 5H,(C- m-Ph3,10), 75.4 mPh2,p-Ph2), (C-2), 75.1 (C- 7.552’), 73.9 (m, ( m-Ph-3,C-13), 72.1 m-Ph1, (C-7), o-Ph2, 58.42 ( 4H),C-8), 7.62 56.3 (tt, (C- 1H,8’-Lipoate), p-Ph1), 7.7752.67 (dd, (C-3 2H,’), 45.6 o-Ph3), (C- 8.153), 43.22 (dd, ( 2H,C-15), o-Ph1). 40.20 ( C-9’-Lipoate), 38.21 (C10’-Lipoate), 35.6 (C-C-14), 34.91, (C4’-Lipoate) 34.5 (C- 6), 28.5 (C-17), 28.22 (C5’-Lipoate), 22.56 (4-OAcMe), 20.9 (10-OAcMe), 14.5 (C-18), 9.95 (C19).

FigureFigure 3. 3.Chemical Chemical structure structure of of the the IDD-1040. IDD-1040. Molecular Molecular formula: formula: CC5555HH6363NO1515SS2.2 . 2.2. 13C-NMR (CDCl ) 2.3. Biological Study Results3 203.5The aim (C-9), of this 172.2 study (C-1), was 171.1 to define (C=O, the OAc-10), maximum 169.9 tolerated (C=O, dose OAc-4), (MTD 168.1) for (C the=O, compound lipoate), 167.0IDD- (C1040,=O, in Ph3), order 167.01 to avoid (C=O, any Ph1), negative 142.8 (C-12), parameters 136.9 associated(q-Ph3), 133.8 with (p-Ph1),133.1 administration, (C11), such 132.7 as (qPh2), severe 130.01adverse (o-Ph1), behavior, 129.1, weight 128.7, loss 127, > 127.7,20% compa 126.5 (Aromaticred to pre- C),administration, 83.2 (C-4), 81.1 and (C-1), even 79.1 death. (C-20), This 76.2 was (C-10), done 75.4to establish (C-2), 75.1 the (C-2’),form of 73.9 clinical (C-13), applicability, 72.1 (C-7), 58.42druggability, (C-8), 56.3 and (C-8’-Lipoate), toxicity of the 52.67 new (C-3’), compound. 45.6 (C-3), The 43.22potential (C-15), acute 40.20 toxicity ( C-9’-Lipoate), of IDD-1040 38.21 was (C10’-Lipoate), assessed following 35.6 (C-C-14), a single, 34.91, slow (C4’-Lipoate)-bolus intravenous 34.5 (C-6), (IV) 28.5injection (C-17), into 28.22 male (C5’-Lipoate), and female BALB/c 22.56 (4-OAcMe), mice. 20.9 (10-OAcMe), 14.5 (C-18), 9.95 (C19). 2.3. BiologicalThe incidence Study of Results mortality was confined to mice injected with IDD-1040 at the highest dosages. It was found that mortality occurred during or immediately following the treatment of one out of one maleThe mouse aim injected of this with study 500 was mg/kg, to define One male the maximummouse injected tolerated with 350 dose mg (MTD)/kg, and for 1/1 the female compound mouse IDD-1040,injected with in order300 mg/kg. to avoid Prior any to negative death, the parameters mice exhibited associated lateral with recumbency administration, and severe such asdyspnea. severe adverseClinical behavior, signs confined weight loss to > mice20% compared injected with to pre-administration, the vehicle control and even(Cremophor death. This EL:ethanol was done toabsolute:physiological establish the form of saline) clinical were applicability, mainly ataxia, druggability, decreased and motor toxicity activity, of the new dyspnea, compound. and lateral The potentialrecumbency. acute toxicity of IDD-1040 was assessed following a single, slow-bolus intravenous (IV) injectionSigns into were male noted and immediately female BALB or/c mice.five minutes after post-injection and lasted up to 30 min. No clinicalThe signs incidence were ofnoted mortality in any was of confinedthe surviving to mice mice injected during with the IDD-1040 14-day period, at the highest but all dosages. exhibited It wasweight found gain, that which mortality was occurredestimated during to be orlower immediately than that following of the vehicle the treatment control ofgroup. one out Furthermore, of one male mouseno gross injected pathological with 500 findings mg/kg, Onewere male noted mouse in any injected of the withmice 350at the mg time/kg, andof their 1/1 female schedul mouseed necropsy, injected with14 days 300 post mg/-kg.injection. Prior toIn death,view of the these mice results exhibited obtained lateral under recumbency the conditions and severe of this dyspnea. study, it Clinicalmay be signsconcluded confined that to the mice MTD injected level of with IDD the-1040 vehicle formulated control (Cremophoras a stable emulsion EL:ethanol in Cremophor absolute:physiological EL:ethanol saline)absolute:physiological were mainly ataxia, saline decreased following motor IV activity,bolus injection dyspnea, over and one lateral minute recumbency. into male or female BALB/cSigns mice were is 250 noted mg/kg. immediately Based on or the five MTD minutes concentration, after post-injection IDD-1040 andwas lastedfurther up studied to 30 min. in an No in clinical signs were noted in any of the surviving mice during the 14-day period, but all exhibited weight gain, which was estimated to be lower than that of the vehicle control group. Furthermore, no gross pathological findings were noted in any of the mice at the time of their scheduled necropsy, 14 days post-injection. In view of these results obtained under the conditions of this study, it may be Int. J. Mol. Sci. 2019, 20, 4965 5 of 15 concluded that the MTD level of IDD-1040 formulated as a stable emulsion in Cremophor EL:ethanol Int.absolute:physiological J. Mol. Sci. 2019, 20, x FOR saline PEERfollowing REVIEW IV bolus injection over one minute into male or female BALB5 of 15/c mice is 250 mg/kg. Based on the MTD concentration, IDD-1040 was further studied in an in vivo vivoxenograft xenograft model model with with a human a human non-small-cell, non-small-cell, lung lung cancer cancer (NSCLC) (NSCLC A549) A549 cell cell line, line, andand thethe data indicate activity inhibiting tumortumor volumevolume (cell(cell growth) growth) in in a a dose-dependent dose-dependent manner, manner, as as seen seen in in Figure Figure4. 4.

Figure 4 4.. TheThe effect eﬀect of IDD IDD-1040-1040 on on tumor tumor volume volume in in an an inin vivo vivo nonnon-small-cell,-small-cell, lung lung cancer cancer ( (NSCLC)NSCLC) xenograft mousemouse model.model. TheThe tumor tumor size size in in all all mice mice treated treated was was measured measured every every other other day, day, starting starting three 3 threeweeks weeks after cell after transplantation, cell transplantation, and when and tumor when size tumor reached size about reached 100 mm about. The 100 IV administration mm3. The IV administrationof vehicle, Taxol of (paclitaxel) vehicle, Taxol 20 mg (paclitaxel)/kg, IDD-1040 20 mg/kg, 125 mg IDD/kg,-1040 and 125 IDD-1040 mg/kg, 250 and mg IDD/kg-1040 was performed250 mg/kg wasweekly performed for four weeksweekly (on for daysfour 23,weeks 30, 37,(on and days 44). 23, The 30, administration37, and 44). The of drugadministration or vehicle of was drug via IVor vehiclebolus injection was via overIV bolus one minute.injection over one minute.

The results clearly show that IDD IDD-1040-1040 profoundly inhibited tumor growth, and the results are statistically significant significant in in comparison comparison to tothe the clinically clinically approved approved PTX, PTX, which which served served as a positive as a positive control controlin the study. in the As study. seen, As tumor seen, growth tumor continuedgrowth continued after PTX after cessation, PTX cessation, confirming confirming that IDD-1040 that IDD exhibited-1040 exhibiteda superior a ability superior to inhibit ability growth to inhibit for twogrowth weeks for after two the weeks treatment after cessation. the treatment To investigate cessation. this To investigateeffect in view this of effectthe MTDs in view of PTX of the(20 mg MTDs/kg) of and PTX IDD-1040 (20 mg/kg) (250 mg and/kg), IDD more-1040 frequent (250 mg/kg), regimens more at frequentlower doses regimens were tested, at lower as doses indicated were in tested, Figure as5. indicated in Figure 5.

Int. J. Mol. Sci. 2019, 20, 4965 6 of 15 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 of 15

Figure 5. The effect of IDD-1040 on tumor volume in an in vivo NSCLC xenograft mouse model, given Figure 5. The effect of IDD-1040 on tumor volume in an in vivo NSCLC xenograft mouse model, given more frequently at lower dosages. Post-IV administration of vehicle (every 4 days for 24 days), Taxol more frequently at lower dosages. Post-IV administration of vehicle (every 4 days for 24 days), Taxol (paclitaxel) (10 mg/kg every four days for 24 days), IDD-1040 (50 mg/kg and 125 mg/kg every four days (paclitaxel) (10 mg/kg every four days for 24 days), IDD-1040 (50 mg/kg and 125 mg/kg every four for 24 days), and IDD-1040 (50 mg/kg once a day for five days, and every two days for 20 days). days for 24 days), and IDD-1040 (50 mg/kg once a day for five days, and every two days for 20 days). Compared to the experiment in Figure6, it should be noted that IDD-1040 at 20% of its MTD (a doseC ofompared 50 mg/kg) to stillthe reducedexperiment tumor in Figure growth 6, significantly it should be whennoted comparedthat IDD-1040 to PTX at 20% at 50% of ofits itsMTD MTD. (a Thisdose wasof 50 seen mg/kg) in a still variety reduc ofed regimens tumor growth employing significantly dosages when ranging compared from 20% to toPTX 100% at 50% of the of MTDits MTD. for IDD-1040,This was seen and in 50% a variety to 100% of of regimens the MTD employing for PTX. This dosages confirms ranging that from IDD-1040 20% hasto 100% a superior of the abilityMTD for to inhibitIDD-1040, tumor and growth, 50% to even100% for of anotherthe MTD two for weeks PTX. afterThis theconfirms treatment that cessationIDD-1040 and has at a 20%superior of the ability MTD, whichto inhibit was tumor not the growth, case for even PTX. forIn another other words,two weeks IDD-1040, after the at treatment both the MTD cessation given and weekly at 20% for of four the weeksMTD, which and at was half thatnot the dose, case was for more PTX. e ffInective other than words, PTX. IDD Moreover,-1040, at the both results the MTD for PTX given and weekly IDD-1040, for givenfour weeks more and frequently, at half eitherthat dose, every was two more or four effective days, than show PTX. that Moreover, IDD-1040 wasthe results more e ffforective PTX thanand PTX.IDD-1040 In addition,, given more there frequently,was a dose-response either every relationship two or four for IDD-1040.days, show All that six IDD IDD-1040-1040 was regimens more thateffective are shown than PTX. in Figures In addition,4 and5 suppressedthere was a tumor dose- growthresponse more relationship than PTX, for with IDD the-1040. two regimensAll six IDD of - 1251040 mg regimens/kg every that four are days shown for in 24 F daysigures (for 4 and a total 5 suppressed of 750 mg /tumorkg) or growth 50 mg/kg more every than other PTX, day with for the 20 daystwo regimens (for a total of of 125 500 mg/kg mg/kg) every providing four days the bestfor 24 responses. days (for Their a total effi ofcacies 750 mg/kg) are statistically or 50 mg/kg significant every whenother comparedday for 20 todays untreated (for a andtotal paclitaxel-treated of 500 mg/kg) providing groups. Thethe pbest-values responses. are less Their than 0.01.efficacies are statisticallyIn order significant to verify thewhen toxicity compared of IDD-1040 to untreated following and IVpaclitaxel administration,-treated groups. animal bodyThe p weight-values andare survivalless than were0.01. monitored throughout the study. In general, a decrease in animal body weight in the rangeIn of order 30% andto verify higher the is toxicity a marker of forIDD toxicity-1040 following (see Figure IV6 administration,). animal body weight and survivalAs canwere be monitor seen, noed marked throughout body the weight study. decrease In general, was observeda decrease during in animal the studybody weight in any ofin the groups;range of in30% fact, and an higher increase is a inmarker animal for body toxicity weight (see Figure was recorded 6). in all groups. This observation supports the toxicity profile of IDD-1040. Tumor growth increases the weight of a mouse. Survival and mortality were also tested, and the results are shown in Figure7.

Int. J. Mol. Sci. 2019, 20, 4965 7 of 15 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 7 of 15

FigureFigure 6. 6Animal. Animal body body weight weight curvescurves followingfollowing the IV IV administration administration of of IDD IDD-1040,-1040, Taxol Taxol (paclitaxel) (paclitaxel),, andand vehicle vehicle in in an anin in vivo vivoA549 A549 NSCLC NSCLC xenograftxenograft study in mice. mice. Animals Animals were were treated treated with with vehicle, vehicle, paclitaxelpaclitaxel (PTX, (PTX at, at 100% 100% of of the the maximum maximum toleratedtolerated dose ( (MTD)),MTD)), and and IDD IDD-1040-1040 (at (at 50% 50% of ofthe the MTD) MTD) Int.administered J. Mol.administered Sci. 2019,weekly 20 weekly, x FOR for PEERfor four four REVIEW weeks. weeks. 8 of 15

As can be seen, no marked body weight decrease was observed during the study in any of the groups; in fact, an increase in animal body weight was recorded in all groups. This observation supports the toxicity profile of IDD-1040. Tumor growth increases the weight of a mouse. Survival and mortality were also tested, and the results are shown in Figure 7.

FigureFigure 7. 7Animal. Animal survival survival and and mortalitymortality curves following the the IV IV administration administration of ofIDD IDD-1040,-1040, Taxol Taxol (paclitaxel),(paclitaxel) and, and vehicle vehicle in in an anin vivoin vivoA549 A549 NSCLC NSCLC xenograft xenograft study study in mice.in mice. Animals Animals were were treated treated with vehicle,with vehicle, PTX (at PTX 100% (at of 100% the MTD),of the MTD), IDD-1040 IDD (at-1040 50% (at of 50% the of MTD), the MTD), andIDD-1040 and IDD-1040 (at 100% (at 100% of the of MTD),the administeredMTD), administered weekly for weekly four for weeks. four weeks.

The survival and mortality of mice was observed even in the vehicle group (11%). As expected, tumor growth reaching a level of >1400 mm3 may cause death in a mouse, especially in the late stage. The following regimens: IDD-1040 250 mg/kg Q7dx4 regimen (100% of the MTD, once a week for four weeks, total of four injections), IDD-1040 125 mg/kg Q4dx6 regimen (20% of the MTD, twice a week for three weeks, total of six injections), and IDD-1040 50 mg/kg Q1dx5 regimen (20% of the MTD, once a day for five days, total of five injections), caused mortality at rates of 54%, 54%, and 22% respectively, which was anticipated due to the amount administered per week to mice that were immune-deficient and bearing tumors to begin with. In the rest of the IDD-1040 treated groups (125 mg/kg Q7dx4, 50 mg/kg Q4dx6, and 50 mg/kg Q2dx10), and in the PTX-treated groups, death was not observed. Based on the survival data presented here, the administration of 150 mg/kg IDD-1040 per week in tumor-bearing mice with severe combined immunodeficiency (SCIDs) can be considered safe. Thus, a strong dependency on the dosing schedule is observed. As PTX use, like that of related anti-mitotic drugs, is generally associated with manageable toxicity, for which neutropenia is among the most common clinical side effects, the difference in toxicity between IDD-1040 and PTX was evaluated. The whole blood of the treated mice was subjected to neutrophil assessment, as indicated in results shown in Figure 8 below.

Int. J. Mol. Sci. 2019, 20, 4965 8 of 15

The survival and mortality of mice was observed even in the vehicle group (11%). As expected, tumor growth reaching a level of >1400 mm3 may cause death in a mouse, especially in the late stage. The following regimens: IDD-1040 250 mg/kg Q7dx4 regimen (100% of the MTD, once a week for four weeks, total of four injections), IDD-1040 125 mg/kg Q4dx6 regimen (20% of the MTD, twice a week for three weeks, total of six injections), and IDD-1040 50 mg/kg Q1dx5 regimen (20% of the MTD, once a day for five days, total of five injections), caused mortality at rates of 54%, 54%, and 22% respectively, which was anticipated due to the amount administered per week to mice that were immune-deficient and bearing tumors to begin with. In the rest of the IDD-1040 treated groups (125 mg/kg Q7dx4, 50 mg/kg Q4dx6, and 50 mg/kg Q2dx10), and in the PTX-treated groups, death was not observed. Based on the survival data presented here, the administration of 150 mg/kg IDD-1040 per week in tumor-bearing mice with severe combined immunodeficiency (SCIDs) can be considered safe. Thus, a strong dependency on the dosing schedule is observed. As PTX use, like that of related anti-mitotic drugs, is generally associated with manageable toxicity, for which neutropenia is among the most common clinical side effects, the difference in toxicity between IDD-1040 and PTX was evaluated. The whole blood of the treated mice was subjected to neutrophilInt. J. Mol. Sci. assessment, 2019, 20, x FOR as PEER indicated REVIEW in results shown in Figure8 below. 9 of 15

FigureFigure 8.8. Assessment of neutropenia in mice treated with vehicle control (blue) and IDD-1040IDD-1040 (green) versusversus PTXPTX (red).(red). The results clearly show that IDD-1040 caused a 28.7% decrease in the neutrophil count compared The results clearly show that IDD-1040 caused a 28.7% decrease in the neutrophil count to the vehicle control, while PTX caused a decrease in the neutrophil count of about 71.34% compared compared to the vehicle control, while PTX caused a decrease in the neutrophil count of about 71.34% to the vehicle control. As can be seen, IDD-1040 induces a minimal level of neutropenia in mice compared to the vehicle control. As can be seen, IDD-1040 induces a minimal level of neutropenia in compared to PTX. mice compared to PTX. Our primary goal in the current study was to introduce a safe and efficient drug candidate for Our primary goal in the current study was to introduce a safe and efficient drug candidate for cancer treatment, while minimizing the adverse side effects commonly seen in PTX-treated patients. cancer treatment, while minimizing the adverse side effects commonly seen in PTX-treated patients. For this purpose, a novel PTX conjugate (IDD-1040) was designed and synthesized, that attains higher For this purpose, a novel PTX conjugate (IDD-1040) was designed and synthesized, that attains higher efficiency and lower toxicity. To conclude, the above results clearly suggest a substantial enlargement efficiency and lower toxicity. To conclude, the above results clearly suggest a substantial enlargement of the therapeutic window for IDD-1040 compared to PTX. IDD-1040 was studied using an in vivo of the therapeutic window for IDD-1040 compared to PTX. IDD-1040 was studied using an in vivo xenograft model in mice injected with NSCLC A549 cells. In that model, when both IDD-1040 and PTX xenograft model in mice injected with NSCLC A549 cells. In that model, when both IDD-1040 and were administered at the MTD, IDD-1040 was far more effective than PTX in suppressing tumor growth. PTX were administered at the MTD, IDD-1040 was far more effective than PTX in suppressing tumor growth. A clear dose-response curve was seen up to the MTD. In a single-dose, intravenous, non-good laboratory practice (non-GLP) toxicity study in mice, the MTD was determined to be 250 mg/kg. The results from the in vivo study clearly show that IDD-1040 inhibited tumor growth more effectively than PTX, which served as a positive control in the study. Specifically, it should be noted that 20% of the MTD for IDD-1040 (50 mg/kg) reduced tumor growth significantly when compared to 50%–100% of the MTD for PTX. This study confirms IDD-1040′s ability to inhibit tumor growth even two weeks after the cessation of treatment. This study in mice conceptually proves and demonstrates the efficacy of IDD-1040 against NSCLC, and thus it is worth developing. In the context of the in vitro study, IDD-1040 shows an absence of cytotoxicity in normal cells (see Figure 9), and greater in vivo efficacy in NSCLC xenograft mice (see Figures 4 and 5). Thus, IDD-1040 appears to function as a pro-drug (slow release system) for PTX and this could explain the improved efficacy and lesser toxicity that are shown in the in vivo model.

Int. J. Mol. Sci. 2019, 20, 4965 9 of 15

A clear dose-response curve was seen up to the MTD. In a single-dose, intravenous, non-good laboratory practice (non-GLP) toxicity study in mice, the MTD was determined to be 250 mg/kg. The results from the in vivo study clearly show that IDD-1040 inhibited tumor growth more effectively than PTX, which served as a positive control in the study. Specifically, it should be noted that 20% of the MTD for IDD-1040 (50 mg/kg) reduced tumor growth significantly when compared to 50–100% of the MTD for PTX. This study confirms IDD-10400s ability to inhibit tumor growth even two weeks after the cessation of treatment. This study in mice conceptually proves and demonstrates the efficacy of IDD-1040 against NSCLC, and thus it is worth developing. In the context of the in vitro study, IDD-1040 shows an absence of cytotoxicity in normal cells (see Figure9), and greater in vivo efficacy in NSCLC xenograft mice (see Figures4 and5). Thus, IDD-1040 appears to function as a pro-drug (slow release system) for PTX and this could explain the improved efficacy and lesser toxicity Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 10 of 15 that are shown in the in vivo model.

Figure 9. Cytotoxicity of PTX (blue) versus IDD-1040 (green). Figure 9. Cytotoxicity of PTX (blue) versus IDD-1040 (green). Proof of concept for IDD-1040 has been established. We have presented the results of an extensive Proof of concept for IDD-1040 has been established. We have presented the results of an in vitro and in vivo evaluation of our lead compound, which emerges as a highly promising agent, extensive in vitro and in vivo evaluation of our lead compound, which emerges as a highly promising certainly worthy of vigorous future development. agent, certainly worthy of vigorous future development. IDD-1040 induces a minimal level of neutropenia in mice compared to Paclitaxel. This result IDD-1040 induces a minimal level of neutropenia in mice compared to Paclitaxel. This result supports our expectation, based on a previously established safety profile, that side effects would be supports our expectation, based on a previously established safety profile, that side effects would be greater in mice treated with PTX than in those treated with IDD-1040. greater in mice treated with PTX than in those treated with IDD-1040. 3. Materials and Methods 3. Materials and Methods The chemicals (paclitaxel, lipoic acid, DCC, DMAP, dry dichloromethane, ethyl acetate, and dichloromethane)The chemicals were (paclitaxel, purchased lipoic from acid, Sigma DCC, (Rehovot, DMAP, Israel).dry dichloromethane, Acetonitrile (ACN), ethyl HPLC-gradeacetate, and solvent,dichloromethane) and water were HPLC-grade purchased were from purchased Sigma ( fromRehovot Biolab, Israel (Jerusalem,). Acetonitrile Israel). (ACN), HPLC-grade solvent, and water HPLC-grade were purchased from Biolab (Jerusalem, Israel). 3.1. Purity of IDD-1040 3.1. Purity of IDD-1040 The purity of IDD-1040 was determined using an HPLC system (Thermo Scientific, San Jose, CA, USA),The which purity included of IDD an-1040 Accela was Pumpdetermined with a using degasser an HPLC module, system an Accela (Thermo autosampler, Scientific, and San anJose, Accela CA, PDAUSA), detector. which included HPLC-grade an Accela acetonitrile Pump was with purchased a degasser from module, J. T. Baker an Accela (Phillipsburg, autosampler, NJ, USA), and and an HPLC-gradeAccela PDA detector.water from HPLC Biolab-grade Ltd. acetonitrile (Jerusalem, was Israel). purchased An HPLC-cartridge from J. T. Baker LiChroCART (Phillipsburg® 250-4, NJ, USA), and HPLC-grade water from Biolab Ltd. (Jerusalem, Israel). An HPLC-cartridge LiChroCART® 250-4 LiChrospher® 100 RP-18 (5μm) (Merck, Roch-Ha’ayin, Israel) and a LiChroCART® 4-4 LiChrospher® 100 RP-18 (5 μm) guard column were used to protect the analytical column.

3.2. Chemical Characterization

3.2.1. 1H-NMR and 13C-NMR Proton and carbon NMR measurements of IDD-1040 were carried out on a Bruker Avance II 500 spectrometer, which was equipped with a 5-mm indirect detection probe with a Z gradient. Positive ESI MS: The molecular mass was determined by HR-ESI–MS (high-resolution electrospray ionization mass spectrometry) in positive mode (LTQ Orbitrap XL hybrid ion trap with a high-resolution Orbitrap detection system, Thermo Scientific, Waltham, MA, USA).

3.2.2. Elemental Analysis

Int. J. Mol. Sci. 2019, 20, 4965 10 of 15

LiChrospher® 100 RP-18 (5 µm) (Merck, Roch-Ha’ayin, Israel) and a LiChroCART® 4-4 LiChrospher® 100 RP-18 (5 µm) guard column were used to protect the analytical column.

3.2. Chemical Characterization

3.2.2. Elemental Analysis Determination of C, H, and N was performed with a Perkin-Elmer 2400 series II Analyzer, which uses a combustion method (950–1000 ◦C) to convert the sample elements to simple gases. The system uses a steady-state, wave front chromatographic approach to separate the controlled gases, which are detected as a function of their thermal conductivity. High-speed microprocessor control, solid-state components, and built-in diagnostics ensure good performance and reliability. The measurement of sulfur percentage was done with the oxygen-ﬂask combustion method and subsequent gravimetric titration and through ion chromatography analysis, using a Dionex IC system.

3.3. Synthesis of IDD-1040 IDD-1040 was synthesized in a one-step reaction. The condensation of PTX with LA using N,N0-dicyclohexylcarbodiimide (DCC)/4-(dimethylamino) pyridine (DMAP) as a coupling activator forms IDD-1040 (see Figure 10). To a dry DCM solution, 1.8 g (8.78 mmol) of lipoic acid and 1.8 g (8.78 mmol) of N,N0-dicyclohexylcarbodiimide (DCC) were added. The mixture was stirred for 15 min. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 11 of 15 N,N0-dicyclohexylurea (DCU) was removed by filtration, and the anhydride was added drop-wise to a solution of 5.0 g (5.85 mmol) of PTX and 700 mg (5.58 mmol) of DMAP in dry dichloromethane. The Determination of C, H, and N was performed with a Perkin-Elmer 2400 series II Analyzer, which resulting mixture was stirred at room temperature with a nitrogen inlet for two hours. The organic uses a combustion method (950–1000 °C) to convert the sample elements to simple gases. The system layer was washed with 10% NaHCO once, 1% HCl twice, and finally, with brine. After drying with uses a steady-state, wave front chromatographic3 approach to separate the controlled gases, which are Na SO , the solution was concentrated with a vacuum. The resulting residue was separated by silica detected2 4 as a function of their thermal conductivity. High-speed microprocessor control, solid-state gel chromatography using DCM/ethyl acetate 7:3 as an eluent (Rf = 0.45) to yield the product (IDD-1040) components, and built-in diagnostics ensure good performance and reliability. The measurement of as a pale-yellow solid (4.2 g, 69%). The IDD-1040 product was further dissolved in dry DCM and left sulfur percentage was done with the oxygen-flask combustion method and subsequent gravimetric at 20 C. Traces of DCU were removed by filtration, and the solution was evaporated, yielding 4.2 g, titration− ◦ and through ion chromatography analysis, using a Dionex IC system. or 69%. The purity of the product was more than 97%, as assessed by HPLC. The product was further 1 13 characterized3.3. Synthesis of by IDDH-NMR,-1040 C-NMR, positive ESI MS, and elemental analyses.

Figure 10. The synthesis pathway of IDD-1040. Figure 10. The synthesis pathway of IDD-1040.

IDD-1040 was synthesized in a one-step reaction. The condensation of PTX with LA using N,N′- dicyclohexylcarbodiimide (DCC)/4-(dimethylamino) pyridine (DMAP) as a coupling activator forms IDD-1040 (see Figure 10). To a dry DCM solution, 1.8 g (8.78 mmol) of lipoic acid and 1.8 g (8.78 mmol) of N,N′-dicyclohexylcarbodiimide (DCC) were added. The mixture was stirred for 15 min. N,N′-dicyclohexylurea (DCU) was removed by filtration, and the anhydride was added drop-wise to a solution of 5.0 g (5.85 mmol) of PTX and 700 mg (5.58 mmol) of DMAP in dry dichloromethane. The resulting mixture was stirred at room temperature with a nitrogen inlet for two hours. The organic layer was washed with 10% NaHCO3 once, 1% HCl twice, and finally, with brine. After drying with Na2SO4, the solution was concentrated with a vacuum. The resulting residue was separated by silica gel chromatography using DCM/ethyl acetate 7:3 as an eluent (Rf = 0.45) to yield the product (IDD- 1040) as a pale-yellow solid (4.2 g, 69%). The IDD-1040 product was further dissolved in dry DCM and left at −20 °C. Traces of DCU were removed by filtration, and the solution was evaporated, yielding 4.2 g, or 69%. The purity of the product was more than 97%, as assessed by HPLC. The product was further characterized by 1H-NMR, 13C-NMR, positive ESI MS, and elemental analyses.

3.4. Drug Candidate Formulation for In-Vivo Experiments IDD 1040 has lower aqueous solubility than Paclitaxel, and as a result, it was formulated as a 6 mg/mL formulation (Taxol®) containing 527 mg Cremophor EL and 49.7% (v/v) dehydrated alcohol. This formulation was diluted to 0.3–1.2 mg/mL before administration. The formulated solution was subjected to a stability study at RT and at 4 °C for 21 days and was found to be stable for at least 24h under both conditions. For the toxicological and in vivo studies presented here, the formulated solution of IDD-1040 was prepared freshly before each use. As well, the control compound (PTX) and vehicle were reconstituted with a solution composed of 14% Cremophor EL, 3.5% ethanol, and 82.5% saline.

Int. J. Mol. Sci. 2019, 20, 4965 11 of 15

3.4. Drug Candidate Formulation for In-Vivo Experiments IDD 1040 has lower aqueous solubility than Paclitaxel, and as a result, it was formulated as a 6 mg/mL formulation (Taxol®) containing 527 mg Cremophor EL and 49.7% (v/v) dehydrated alcohol. This formulation was diluted to 0.3–1.2 mg/mL before administration. The formulated solution was subjected to a stability study at RT and at 4 ◦C for 21 days and was found to be stable for at least 24 h under both conditions. For the toxicological and in vivo studies presented here, the formulated solution of IDD-1040 was prepared freshly before each use. As well, the control compound (PTX) and vehicle were reconstituted with a solution composed of 14% Cremophor EL, 3.5% ethanol, and 82.5% saline.

3.5. Animals Two types of animal were employed in the current study: ten-week-old male and female BALB/c mice were used to carry out the experiments for MTD determination, and ten-week-old male and female NOD SCID mice were employed to investigate the drug’s effect on subcutaneous tumor growth, body weight, and neutropenia. The animals were obtained from Harlan biotech (Rehovot, Israel). They had unrestricted access to food and water, were housed in temperature and humidity-controlled rooms, and were kept on a 12-h light/dark cycle. The testing procedure was based on guidelines established by internationally accepted guidance. To confirm whether IDD-1040 has the potential to serve as a superior anti-cancer agent, a xenograft model was employed to assess the efficacy of IDD-1040 (monotherapy) versus PTX. The study design used the following parameters: an A549 human NSCLC cell line, 7–9 SCID mice per group, and an initial tumor size of ~80–100 mm3. Both drugs were administered intravenously (IV) and were given in different regimens, as indicated below. There were nine test groups, with the following regimens: (1) vehicle (14% Cremophor EL, 3.5% ethanol, and 82.5% saline), (2) IDD-1040 125 mg/kg Q7dx4 (50% MTD, once a week, for a total of four injections); (3) IDD-1040 250 mg/kg Q7dx4 (100% MTD, once a week, total of four injections); (4) IDD-1040 50 mg/kg Q4dx6 (20% MTD, two times a week, total of six injections); (5) IDD-1040 125 mg/kg Q4dx6 (20% MTD, two times a week, total of six injections); (6) IDD-1040 50 mg/kg Q1dx5 (20% MTD, once a day, total of five injections); (7) IDD-1040 50 mg/kg Q2dx10 (20% MTD, three times a week, total of ten injections); (8) PTX 20 mg/kg Q7dx4 (100% MTD, once a week, total of four injections); and (9) PTX 10 mg/kg Q4dx6 (50% MTD, two times a week, total of six injections). The dosage volume in each injection was 10 mL/kg. This study was performed following an application-form review by the National Council for Animal Experimentation and after receiving their approval (number IL-10-5-52, 11 March 2012).

3.6. Study Design The procedure for establishing the MTD utilized eight groups of mice, each consisting of three males and three females. Two-hundred ﬁfty microliters of solution (IDD-1040 solution or blank solution) were injected in a single, slow-bolus intravenous (IV) injection into male and female BALB/c mice, in the form of a stable emulsion, over one minute, at seven doses of 100, 150, 175, 250, 300, 350, and 500 mg/kg. An additional two groups of three males and three females were injected with the vehicle control item (i.e., Cremophor EL: ethanol absolute: physiological saline), and PTX 20 mg/kg, and served as negative and positive control groups, respectively. All of the surviving mice were monitored for 14 days.

3.7. Toxicology A non-GLP, single-dose, intravenous toxicity study was conducted on BALB/c mice. IDD-1040 doses of Cremophor® EL: ethanol absolute: physiological saline solution at 0, 100, 150, 175, 250, 300, 350, and 500 mg/kg were administered as one-minute injections, and the animals were monitored for 14 days. Groups of three of each sex were administered doses between 0 and 250 mg/kg, whereas Int. J. Mol. Sci. 2019, 20, 4965 12 of 15 at doses of 300, 350 and 500 mg/kg only one animal (male or female) was treated. The parameters evaluated included clinical signs, body weight, and gross pathology. Mortality occurred at 300 mg/kg and above; all the animals in these groups died immediately post-dose. In these animals, lateral recumbency and severe dyspnea were observed. All IDD-1040-treated animals exhibited weight gain over the 14-day observation period, but the gain was lower than for the vehicle controls. No gross pathological ﬁndings were observed at necropsy in any animals. Based on these data, the single MTD was concluded to be 250 mg/kg.

3.8. Cell Culture

3.8.1. Cancer Cells Cell from the human, non-small-cell, lung cancer (NSCLC) cell line A549 were purchased from the American Type Culture Collection (Manassas, VA, USA) ATCC. This cell line was cultured in RPMI 1640 media (Biological Industry, Ltd., Beit Haemek, Israel), together with 1 mM of l-glutamine, 25 mM of HEPES buﬀer, 100 U/mL of penicillin, 100 ng/mL of streptomycin, and 10% fetal calf serum (FCS) (RPMI complete media), in an environment of 95% air and 5% CO2 at 37 ◦C.

3.8.2. Normal Cells Human umbilical vein/vascular endothelial cells (HUVECs) were purchased from the American Type Culture Collection (Manassas, VA, USA) ATCC. Endothelial cells naturally constitute the monolayer that covers the interior surface of blood vessels, and are most susceptible to circulating drugs. Thus, these cells were chosen to be investigated in vitro with IDD-1040 and PTX. A total of 70,000 cells were grown onto a 6-well-plate and after three-hour’s of cell adherence; IDD-1040 and PTX were added and the plate was incubated for 48 h in an environment of 95% air and 5% CO2 at 37 ◦C. Cells were then detached by trypsin and counted by Guava ViaCount (Guava Technologies, Millipore, Hayward, California, USA) application, a rapid and reliable method for determining cell count and viability.

3.9. Tumor Growth and Size Measurement in a Xenograft Model The NOD SCID mice xenograft model was established using mice aged 8–10 weeks and weighing 20 g. The mice were studied under pathogen-free conditions within the animal house under a 12-h light/12-h dark cycle and with an ad libitum supply of standard chow often used for rodents. Cells from the lung cancer cell line A549 (NSCLC) (5 106 cells/0.2 mL) were injected into the subcutaneous tissue × of the dorsal area of each mouse, and tumors were allowed to grow to approximately 100 mm3 in size. Tumor volume was calculated using the following equation: tumor volume = (π/6)(width)2 length, where volume is measured in cubic millimeters, and width and length are measured in millimeters. The tumor size was measured three weeks after cell transplantation. The treated groups (7–9 animals per group) were mainly groups receiving vehicle, IDD-1040 (50–250 mg/kg), or Taxol (Paclitaxel) (10–20 mg/kg) according to various schedules.

3.10. Neutropenia The control groups were treated with vehicle at 10 mL/kg (Group 1), with PTX at 20 mg/kg (Group 2), and with IDD-1040 at 50 mg/kg (Group 3). The administration of compounds was performed intravenously on a Q1dx5 schedule. Since both paclitaxel and IDD-1040 at the above dosages are well tolerated based on adjusted body weight (ABW, g), and showed similar eﬃcacy against the NSCLC animal model, a daily dosing schedule was used to evaluate the toxicity potentially induced by greater compound exposure over a short period of time. Animals were monitored daily for clinical signs. On the seventh day after the initiation of therapy, all the animals were bled. Whole blood was collected into heparin-containing tubes for hematological examination and neutrophil assessment. No mortality or severe clinical signs were observed in the groups during the study. Int. J. Mol. Sci. 2019, 20, 4965 13 of 15

3.11. Statistical Analysis Data were analyzed using the data analysis module of excel spreadsheet software (v16.0, Microsoft, Redmond, WA, USA). Differences between the control (untreated) and treatment groups were evaluated by applying one-way analysis of variance (ANOVA). A p-value of less than 0.05 was considered statistically significant. The figures display the means and standard deviations.

4. Conclusions Our study shows that the anti-tumor activity of IDD-1040 chemical conjugate chemotherapy in the xenograft animal model is far more favorable than that of PTX. IDD-1040 has shown a much higher MTD (>10-fold) with superior tumor inhibition. Its eﬀect on tumor growth persists even after the cessation of treatment associated with a low-toxicity proﬁle, as well as with a lower mortality rate in mice test groups. All of the results herein demonstrate that IDD-1040 has great potential as a drug candidate for cancer treatment, and hence, is recommended for advancement into clinical trials.

Author Contributions: A.R. conceived the study, raised the funds, and performed the analysis. M.F., and M.R., carried out the statistical analysis of the data. M.R., and A.R. interpreted the data and wrote up the conclusions. M.F., and A.R., wrote the first draft of the manuscript. A.R. edited the manuscript. Funding: This work was partially supported by the Israel Innovation Authority (an incubator project that was granted to IDD Therapeutics, Ltd., between the years 2010 and 2014), and the Israel Cancer Association (R.A.) (grant numbers 20191644 and 20180071). Conflicts of Interest: A.R. is the founder and CEO of IDD Therapeutics, Ltd. The IP owner of IDD-1040 is IDD Therapeutics. We declare that the funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The other two authors declare no conflicts of interest.

References

1. Rowinsky, E.K.; Cazenave, L.A.; Donehower, R.C. Taxol: A novel investigational antimicrotubule agent. J. Natl. Cancer Inst. 1990, 82, 1247–1259. [CrossRef] 2. Peng, L.; Schorzman, A.N.; Ma, P.; Madden, A.J.; Zamboni, W.C.; Benhabbour, S.R.; Mumper, R.J. 2’-(2-bromohexadecanoyl)-paclitaxel conjugate nanoparticles for the treatment of non-small cell lung cancer in an orthotopic xenograft mouse model. Int. J. Nanomed. 2014, 9, 3601–3610. 3. Mabuchi, S.; Isohashi, F.; Yokoi, T.; Takemura, M.; Yoshino, K.; Shiki, Y.; Ito, K.; Enomoto, T.; Ogawa, K.; Kimura, T. A phase II study of postoperative concurrent carboplatin and paclitaxel combined with intensity-modulated pelvic radiotherapy followed by consolidation chemotherapy in surgically treated cervical cancer patients with positive pelvic lymph nodes. Gynecol. Oncol. 2016, 141, 240–246. [CrossRef] [PubMed] 4. Chen, T.; Gong, T.; Zhao, T.; Liu, X.; Fu, Y.; Zhang, Z.; Gong, T. Paclitaxel loaded phospholipid-based gel as a drug delivery system for local treatment of glioma. Int. J. Pharm. 2017, 528, 127–132. [CrossRef] 5. Cohn, D.E.; Sill, M.W.; Walker, J.L.; O’Malley, D.; Nagel, C.I.; Rutledge, T.L.; Bradley, W.; Richardson, D.L.; Moxley, K.M.; Aghajanian, C. Randomized phase IIB evaluation of weekly paclitaxel versus weekly paclitaxel with oncolytic reovirus (Reolysin(R)) in recurrent ovarian, tubal, or peritoneal cancer: An NRG Oncology/Gynecologic Oncology Group study. Gynecol. Oncol. 2017, 146, 477–483. [CrossRef][PubMed] 6. Schettini, F.; Giuliano, M.; De Placido, S.; Arpino, G. Nab-paclitaxel for the treatment of triple-negative breast cancer: Rationale, clinical data and future perspectives. Cancer Treat. Rev. 2016, 50, 129–141. [CrossRef] [PubMed] 7. He, Z.; Schulz, A.; Wan, X.; Seitz, J.; Bludau, H.; Alakhova, D.Y.; Darr, D.B.; Perou, C.M.; Jordan, R.; Ojima, I.; et al. Poly(2-oxazoline) based micelles with high capacity for 3rd generation taxoids: Preparation, in vitro and in vivo evaluation. J. Control. Release 2015, 208, 67–75. [CrossRef] 8. Dumontet, C.; Sikic, B.I. Mechanisms of action of and resistance to antitubulin agents: Microtubule dynamics, drug transport, and cell death. J. Clin. Oncol. 1999, 17, 1061–1070. [CrossRef][PubMed] 9. Jordan, M.A.; Wilson, L. Microtubules as a target for anticancer drugs. Nat. Rev. Cancer 2004, 4, 253–265. [CrossRef] Int. J. Mol. Sci. 2019, 20, 4965 14 of 15

10. Magnani, M.; Maccari, G.; Andreu, J.M.; Diaz, J.F.; Botta, M. Possible binding site for paclitaxel at microtubule pores. FEBS J. 2009, 276, 2701–2712. [CrossRef] 11. Marchetti, P.; Urien, S.; Cappellini, G.A.; Ronzino, G.; Ficorella, C. Weekly administration of paclitaxel: Theoretical and clinical basis. Crit. Rev. Oncol Hematol 2002, 44, S3–S13. [CrossRef] 12. Mollinedo, F.; Gajate, C. Microtubules, microtubule-interfering agents and apoptosis. Apoptosis 2003, 8, 413–450. [CrossRef][PubMed] 13. Pasquier, E.; Kavallaris, M. Microtubules: A dynamic target in cancer therapy. IUBMB Life 2008, 60, 165–170. [CrossRef] 14. Haldar, S.; Chintapalli, J.; Croce, C.M. Taxol induces bcl-2 phosphorylation and death of prostate cancer cells. Cancer Res. 1996, 56, 1253–1255. 15. Carbognin, L.; Sperduti, I.; Nortilli, R.; Brunelli, M.; Vicentini, C.; Pellini, F.; Pollini, G.P.; Giannarelli, D.; Tortora, G.; Bria, E. Balancing activity and tolerability of neoadjuvant paclitaxel- and docetaxel-based chemotherapy for HER2-positive early stage breast cancer: Sensitivity analysis of randomized trials. Cancer Treat. Rev. 2015, 41, 262–270. [CrossRef][PubMed] 16. Tang, S.; Yin, Q.; Su, J.; Sun, H.; Meng, Q.; Chen, Y.; Chen, L.; Huang, Y.; Gu, W.; Xu, M.; et al. Inhibition of metastasis and growth of breast cancer by pH-sensitive poly (beta-amino ester) nanoparticles co-delivering two siRNA and paclitaxel. Biomaterials 2015, 48, 1–15. [CrossRef][PubMed] 17. Rivera, E.; Cianfrocca, M. Overview of neuropathy associated with taxanes for the treatment of metastatic breast cancer. Cancer Chemother. Pharm. 2015, 75, 659–670. [CrossRef][PubMed] 18. Socinski, M.A. Single-agent paclitaxel in the treatment of advanced non-small cell lung cancer. Oncologist 1999, 4, 408–416. [PubMed] 19. Wani, M.C.; Taylor, H.L.; Wall, M.E.; Coggon, P.; McPhail, A.T. Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J. Am. Chem. Soc. 1971, 93, 2325–2327. [CrossRef][PubMed] 20. Hadzic, T.; Aykin-Burns, N.; Zhu, Y.; Coleman, M.C.; Leick, K.; Jacobson, G.M.; Spitz, D.R. Paclitaxel combined with inhibitors of glucose and hydroperoxide metabolism enhances breast cancer cell killing via H2O2-mediated oxidative stress. Free Radic. Biol. Med. 2010, 48, 1024–1033. [CrossRef][PubMed] 21. Hausheer, F.H.; Schilsky, R.L.; Bain, S.; Berghorn, E.J.; Lieberman, F. Diagnosis, management, and evaluation of chemotherapy-induced peripheral neuropathy. Semin. Oncol. 2006, 33, 15–49. [CrossRef][PubMed] 22. Ricciardi, G.R.; Russo, A.; Franchina, T.; Ferraro, G.; Adamo, V. Efficacy of nab-paclitaxel plus trastuzumab in a long-surviving heavily pretreated HER2-positive breast cancer patient with brain metastases. Oncol. Targets 2015, 8, 289–294. 23. Agathos, E.; Tentolouris, A.; Eleftheriadou, I.; Katsaouni, P.; Nemtzas, I.; Petrou, A.; Papanikolaou, C.; Tentolouris, N. Effect of alpha-lipoic acid on symptoms and quality of life in patients with painful diabetic neuropathy. J. Int. Med. Res. 2018, 46, 1779–1790. [CrossRef][PubMed] 24. Kuban-Jankowska, A.; Gorska-Ponikowska, M.; Wozniak, M. Lipoic Acid Decreases the Viability of Breast Cancer Cells and Activity of PTP1B and SHP2. Anticancer Res. 2017, 37, 2893–2898. [PubMed] 25. Li, B.J.; Hao, X.Y.; Ren, G.H.; Gong, Y. Effect of lipoic acid combined with paclitaxel on breast cancer cells. Genet. Mol. Res. 2015, 14, 17934–17940. [CrossRef][PubMed] 26. Whiteman, M.; Tritschler, H.; Halliwell, B. Protection against peroxynitrite-dependent tyrosine nitration and alpha 1-antiproteinase inactivation by oxidized and reduced lipoic acid. FEBS Lett. 1996, 379, 74–76. [CrossRef] 27. Packer, L. alpha-Lipoic acid: A metabolic antioxidant which regulates NF-kappa B signal transduction and protects against oxidative injury. Drug Metab Rev. 1998, 30, 245–275. [CrossRef][PubMed] 28. Wollin, S.D.; Jones, P.J. Alpha-lipoic acid and cardiovascular disease. J. Nutr. 2003, 133, 3327–3330. [CrossRef] 29. Wang, X.; Yu, Y.; Ji, L.; Liang, X.; Zhang, T.; Hai, C.X. Alpha-lipoic acid protects against myocardial ischemia/reperfusion injury via multiple target effects. Food Chem. Toxicol. 2011, 49, 2750–2757. [CrossRef] 30. Ambrosi, N.; Guerrieri, D.; Caro, F.; Sanchez, F.; Haeublein, G.; Casadei, D.; Incardona, C.; Chuluyan, E. Alpha Lipoic Acid: A Therapeutic Strategy that tend to Limit the Action of Free Radicals in Transplantation. Int. J. Mol. Sci. 2018, 19, 102. [CrossRef] 31. Papanas, N.; Ziegler, D. Efficacy of alpha-lipoic acid in diabetic neuropathy. Expert Opin. Pharm. 2014, 15, 2721–2731. [CrossRef][PubMed] Int. J. Mol. Sci. 2019, 20, 4965 15 of 15

32. Ziegler, D.; Low, P.A.; Litchy, W.J.; Boulton, A.J.; Vinik, A.I.; Freeman, R.; Samigullin, R.; Tritschler, H.; Munzel, U.; Maus, J.; et al. Eﬃcacy and safety of antioxidant treatment with alpha-lipoic acid over 4 years in diabetic polyneuropathy: The NATHAN 1 trial. Diabetes Care 2011, 34, 2054–2060. [CrossRef][PubMed] 33. Ziegler, D.; Hanefeld, M.; Ruhnau, K.J.; Hasche, H.; Lobisch, M.; Schutte, K.; Kerum, G.; Malessa, R. Treatment of symptomatic diabetic polyneuropathy with the antioxidant alpha-lipoic acid: A 7-month multicenter randomized controlled trial (ALADIN III Study). ALADIN III Study Group. Alpha-Lipoic Acid in Diabetic Neuropathy. Diabetes Care 1999, 22, 1296–1301. [CrossRef][PubMed] 34. Carlson, D.A.; Smith, A.R.; Fischer, S.J.; Young, K.L.; Packer, L. The plasma pharmacokinetics of R-(+)-lipoic acid administered as sodium R-(+)-lipoate to healthy human subjects. Altern Med. Rev. 2007, 12, 343–351. 35. Bingham, P.M.; Stuart, S.D.; Zachar, Z. Lipoic acid and lipoic acid analogs in cancer metabolism and chemotherapy. Expert Rev. Clin. Pharm. 2014, 7, 837–846. [CrossRef][PubMed] 36. Damnjanovic, I.; Kocic, G.; Najman, S.; Stojanovic, S.; Stojanovic, D.; Veljkovic, A.; Conic, I.; Langerholc, T.; Pesic, S. Chemopreventive potential of alpha lipoic acid in the treatment of colon and cervix cancer cell lines. Bratisl. Lek. Listy 2014, 115, 611–616. [CrossRef][PubMed] 37. Yang, L.; Wen, Y.; Lv, G.; Lin, Y.; Tang, J.; Lu, J.; Zhang, M.; Liu, W.; Sun, X. alpha-Lipoic acid inhibits human lung cancer cell proliferation through Grb2-mediated EGFR downregulation. Biochem. Biophys. Res. Commun. 2017, 494, 325–331. [CrossRef][PubMed] 38. Kismali, G.; Yurdakok-Dikmen, B.; Kuzukiran, O.; Arslan, P.; Filazi, A. Phthalate induced toxicity in prostate cancer cell lines and eﬀects of alpha lipoic acid. Bratisl. Lek. Listy 2017, 118, 460–466. [CrossRef][PubMed]

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).