Polymer complex of WR 2721. Synthesis and radioprotective efficiency

European Journal of Pharmaceutical Sciences 65 (2014) 9–14

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European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier .com/ locate/e jps

Polymer complex of WR 2721. Synthesis and radioprotective efficiency

http://dx.doi.org/10.1016/j.ejps.2014.08.0060928-0987/� 2014 Elsevier B.V. All rights reserved.

⇑ Corresponding authors. Address: Dept. Experimental Radiation Oncology, M.D.Anderson Cancer Center, 1515 Holcombe Blvd., Unit 066, Houston, TX 77030-4009,USA. Tel.: +1 713 792 4860 (K. Mason). Institute of Polymers, Bulgarian Academy ofSciences, Acad. Georgi Bonchev Str., Bl. 103A, Sofia 1113, Bulgaria. Tel.: +359 29792203 (K. Troev).

E-mail addresses: [email protected] (K. Mason), [email protected](K. Troev).

1 These authors contributed equally.

Neli Koseva a, Ivelina Tsacheva a, Violeta Mitova a, Elitsa Vodenicharova a, Jessica Molkentine b,Kathy Mason b,1,⇑, Kolio Troev a,1,⇑a Institute of Polymers, Bulgarian Academy of Sciences, Acad. Georgi Bonchev Str., Bl. 103A, Sofia 1113, Bulgariab M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Unit 066, Houston, TX 77030-4009, USA

a r t i c l e i n f o

Article history:Received 17 April 2014Received in revised form 7 August 2014Accepted 15 August 2014Available online 23 August 2014

Keywords:Poly(hydroxyoxyethylene phosphate)WR 2721Polymer complexRadioprotectorProtection factor

a b s t r a c t

Polymer complex constructed from WR 2721 and poly(hydroxyoxyethylene phosphate) was synthesized.The structure of complex formed was elucidated by 1H-, 13C, 31P NMR and FT-IR spectroscopy. The radio-protector was immobilized via ionic bonds. Radioprotective efficacy was evaluated by clonal survival ofstem cells in crypts of mouse small intestine, and incidence and latency of the acute radiation inducedbone marrow syndrome. Protection factors were assessed for WR 2721 and for the polymer complex. Pro-tection factors for the polymer complex ranged from 2.6 for intestinal stem cell survival to 1.35 for 30 daysurvival (LD50) following whole body radiation exposure. In all cases, the polymer complex was a signif-icantly better radiation protector than the parent compound.

� 2014 Elsevier B.V. All rights reserved.

1. Introduction

The research interest in amifostine (WR 2721) continues longafter its discovery due to its potential role in reducing the biolog-ical effects of ionizing radiation, including lethality, mutagenicity,and carcinogenicity. WR 2721 demonstrated some potential foruse as an emergency whole-body radioprotector as well as foruse with radiotherapy (Brown et al., 1988). Several studies havedemonstrated that amifostine protects normal tissue from bothacute and late radiation damage without protecting the tumor,i.e. amifostine is a selective cytoprotector of normal tissues(Burkon et al., 2003; Koukourakis, 2003; Wasserman, 1999;Wasserman and Brizel, 2001). Maximum radioprotection by ami-fostine is observed when the drug is administered intravenouslyby a 15 min infusion starting 30–60 min before irradiation(Bukowski, 1996). It is shown that its side-effects, such as hypoten-sion, nausea, and vomiting are significantly augmented upon intra-venous administration (Bonner and Shaw, 2002; Cassatt et al.,2002; Schuchter et al., 2002).

A promising approach to improve some characteristics of lowmolecular weight drugs, already approved and used in practice,as well as to impart new valuable properties is the macromolecularapproach, i.e. application of appropriate polymers for drug immo-bilization, chemically conjugated or physically bound to a polymerchain. Polymer chemistry has contributed in various ways to thepresent progress in biology, biochemistry, medicine and pharmacy,providing new highly specific materials. Synthetic polymer formu-lations are becoming more and more attractive as delivery vehiclesbecause of the great flexibility regarding: (i) the type and size ofthe bioactive molecules/agents delivered, (ii) the degree of carrierloading and (iii) the immobilization techniques applied (Duncan,1992; Hoste et al., 2004; Ottenbrite et al., 1978; Rihova et al.,2001; Ringsdorf, 1975; Uhrich, 1997; Uhrich et al., 1999). The bio-degradable, biocompatible and low toxic polyphosphoesters – afamily of polymers including poly(alkylene H-phosphonate)s andderived from them polyphosphates and polyphosphoamidates –are very promising polymers for drug and gene delivery (Tseviet al., 1993; Georgieva et al., 2002; Huang et al., 2004; Jianget al., 2007; Troev et al., 2010, 2007; Pencheva et al., 2008; Zhaoet al., 2003). Polyphosphoesters have the following advantages(Troev, 2012): (i) relative ease of preparation from commerciallyavailable reagents; (ii) the possibility to be constructed from non-toxic and water soluble blocks; (iii) possibility to control thehydrophilic/hydrophobic balance; (iv) relatively narrow molecularweight distributions; (v) the drug-loading capacity is not limited tothe end groups; (vi) the reactive PAH group in repeating unitsallows chemical modification, as well as drug conjugation under

10 N. Koseva et al. / European Journal of Pharmaceutical Sciences 65 (2014) 9–14

mild reaction conditions; (vii) the presence of highly polar P@Ogroups affords possibility for physical immobilization of drugs;(viii) these polymers can be used to prepare gels; (ix) they canbe administered over a wider molecular weight range becausethe PEG blocks will be safely excreted after hydrolysis – importantcharacteristics for intravenous administration; (x) easy to prepareon industrial scale.

The goal of the present study is to evaluate the radioprotectiveefficiency of a newly synthesized polymer complex composed ofpoly(hydroxyoxyethylene phosphate) and WR 2721.

2. Material and methods

2.1. Synthetic procedures

2.1.1. MaterialsPEG with number average molecular weight 600 g.mol-1 (PEG

600) was purchased from Fluka. It was dried prior to use by an aze-otropic distillation with toluene and a subsequent 4 h heating at120 �C under dynamic vacuum. Dimethyl hydrogen-phosphonate(Fluka) and triethylamine (Flika) were distilled prior to use. 2-[(3-aminopropyl)amino]ethane-1-thiol was obtained from Fluka,while hydrobromic acid (48%), 1,3-diaminopropane (99%) and eth-ylene sulfide (98%) were Aldrich products. Acetonitrile (Fluka) wasdried and distilled before use. Trichloroisocyanuric, 97% (SigmaAldrich) was used as received. Dowex 50 was purchased fromFluka.

2.1.2. InstrumentationAll 1H, 31P and 13C NMR spectra were measured on a Bruker

250 MHz spectrometer in D2O or CDCl3 solutions. The infra-red(IR) spectra were recorded on Bruker-Vector 22 FT-IR spectropho-tometer in KBr tablets. SEC measurements were performed on aWaters 244 line equipped with four Ultrastyragel columns withpore sizes 100, 100, 500, and 500 Å and tetrahydrofuran as the car-rier solvent. The molecular weights were calculated using a con-ventional calibration with PEG standards.

2.1.3. Synthesis of poly(oxyethylene hydrogen phosphonate)Poly(oxyethylene hydrogen phosphonate) (POEHP) was

obtained from commercial dimethyl hydrogen phosphonate(DMP) (5.50 g, 0.050 mol) and PEG 600 (19.80 g, 0.033 mol) in atwo stage reaction. The entire synthesis was carried out in a vac-uum distillation set-up. A typical synthetic procedure is describedbellow: In the first low temperature transesterification stage, thetemperature was slowly increased to 135 �C in N2 at atmosphericpressure. The reaction was kept at these conditions for 5 h and ter-minated after the temperature of the methanol vapors droppeddown and its distillation visibly stopped. The second polyconden-sation stage was performed under vacuum of 1 mmHg at elevatedtemperatures for a total of 4.15 h (4 h at 160 �C, 15 min at 185 �C).The product obtained was wax material.

1H NMR (CDCl3), d (ppm): 6.93 (d, 1J(P,H) = 715.87 Hz, ACH2

OP(O)(H)OCH2A); 6.86 (d, 1J(P,H) = 708.61 Hz, CH3OP(O)(H)OA);6.79 (d, 1J(P,H) = 689.10 Hz, HOP(O)(H)OCH2A); 4.14–4.31 (m,AOP(O)(H)OCH2) and 3.60–3.69 (m, AOCH2CH2OA).

13C{H} NMR (CDCl3), d (ppm): 64.70 (d, 2J(P,C) = 5.79 Hz, ACH2

OP(O)(H)OCH2A); 70.18 (d, 3J(P,C) = 5.79 Hz, AOP(O)(H)OCH2CH2A)and 70.58 (s, AOCH2CH2OA).

31P NMR (CDCl3), d (ppm): 11.21 (d sextet, 1J(P,H) = 708.37 Hz,3J(P,H) = 11.74 Hz, ACH2OP(O)(H)OCH3); 10.51 (d quintet;1J(P,H) = 715.46 Hz and 3J(P,H) = 9.82 Hz, ACH2OP(O)(H)OCH2A);and 7.58 (dt; 1J(P,H) = 693.3 Hz, 3J(P,H) = 10.32 Hz,ACH2OP(O)(H)OH).

31P{H} NMR (CDCl3) d (ppm): 11.21 (5.4%); 10.51 (90.1%); 7.58(4.5%).

2.1.4. Synthesis of poly(hydroxyoxyethylene phosphate)2.1.4.1. Synthesis of poly(oxyethylene chlorophosphate). Poly(oxy-ethylene chlorophosphate) (POEClP) was obtained from POEHPand trichloroisocyanuric acid. The entire synthesis was carriedout under inert atmosphere. To a stirred solution of POEHP(2.69 g, 4.17 mmol) in acetonitrile (9 ml) at room temperaturewas added in one portion a solution of trichloroisocyanuric acid(0.333 g, 1.39 mmol) in acetonitrile 13.5 ml. The reaction mixturewas kept for 1 h. Isocyanuric acid was removed from the solutionby filtration.

2.1.4.2. Synthesis of poly(hydroxyoxyethylene phosphate). The syn-thesis of poly(hydroxyoxyethylene phosphate) (PHOEP) was car-ried out under inert atmosphere. A typical synthetic procedure isdescribed bellow: To the stirred solution of POEClP in acetonitrileat room temperature water (0.075 g, 4.17 mmol) and triethylamine(0.297 g, 0.41 ml, 4.17 mmol) were added in one portion. The reac-tion mixture was kept for 30 min. The solution was refrigerated at�12 �C. Triethylamine hydrochloride crystals were removed by fil-tration. The solvent was evaporated and residue was dissolved inwater and was passed through ion exchange resin Dowex 50. Afterdialysis against deionized water the reaction product was freeze-dried. Yield 80%. The structure of PHOEP was proved by NMRspectroscopy.

1H NMR (D2O), d (ppm): 4.00–3.86 (m, CH2OP(O)(OH)OCH2),3.60–3.50 (m, CH2OCH2); 7.18 ppm, (dt, 1J(P,H) = 650.12 Hz, PAH)

13C{H}NMR (D2O), d (ppm): 70.13 (d, 3J(P,C) = 8.2 Hz, POCH2

CH2), 69.59 (CH2OCH2), 64.68 (d, 2J(P,C) = 6.2 Hz, POCH2CH2);31P NMR (D2O), d (ppm): 7.62, d, 1J(P,H) = 653.12 Hz; 2.17 and

0.90 (phosphate structures).31P{H}NMR (D2O), d (ppm): 7.62 (1.35%); 2.17 (3.55%) and 0.90

(95.10%);IR (KBr): 1291 cm�1 b 1259 cm�1 m(P@O), 1102 cm�1 m(H2CAOA

CH2), 1036 cm�1 m (PAOCH2).

2.1.5. Synthesis of S-2-(3-aminopropylamino) ethylphosphorothioicacid dihydrate (amifostine, WR 2721)

Amifostine was synthesized in two stages following a previ-ously described procedure (Piper and Johnston, 1975). Yield:77%; m.p. 145 �C. Purity > 98%.

1H NMR (D2O), d (ppm): 3.39 (t, 3J(H,H) = 5.4 Hz, 2H, CH2CH2

NH-); 3.10–3.18 (m, 4H, H2NCH2CH2CH2NHCH2); 3.04–2.93 (m,2H, CH2CH2S); 2.12 (quintet, 3J(H,H) = 7.8 Hz, 2H,CH2CH2CH2NH);

13C{H} NMR (D2O), d (ppm): 53.05 (CH2NH), 47.27 (NH2CH2),39.41 (NHCH2), 28.40 (d, 2J(P,C) = 2.8 Hz, CH2SP), 26.69(CH2CH2CH2);

31P NMR (D2O), d (ppm): 15.77 (t, 3J(P,H) = 14.09 Hz);IR (KBr): (cm�1): 3471 – NH2 (stretching); 3335 – NH (stretch-

ing); 2922–2742 – CH2 (stretching); 2560 – POH (stretching); 1188– P@O (stretching).

2.1.6. Immobilization of WR 2721 on PHOEPA solution of WR 2721 (0.40 g, 1.6 mmol) in 5 ml water was

added to an aqueous solution of PHOEP (1.06 g, 1.6 mmol) in20 ml distilled water and the mixture was freeze-dried.

31P{H} NMR (D2O), d (ppm): 15.87 (PASCH2); 1.88 (CH2OP(�O)(O)OH); 0.66 (CH2OP(�O)(O) OCH2);

IR (KBr): (cm�1): 3660–3200 – NH2 and NH (stretching); 2887 –CH2 (stretching); 2677–2495 – POH (stretching), 1643 – NH2 andNH (bending); 1243 – P@O (stretching); 1101 – PAOAC, CANand CAOAC (stretching).

PHOEPWR 2721

_O

OH n

_O

O

n_

H3N(CH2)3NH(CH2)2SP(O)(OH)2.2H2O+

H2N(CH2)3NH(CH2)2SP(O)(OH)2.2H2O+

Polymer complex of WR 2721

P-O(CH2CH2O)13

P-O(CH2CH2O)13

Scheme 2. Immobilization of WR 2721 onto poly(hydroxyoxyethylene phosphate).

N. Koseva et al. / European Journal of Pharmaceutical Sciences 65 (2014) 9–14 11

2.2. Biology

2.2.1. MiceMale C3Hf/KamLaw mice were bred and maintained in a spe-

cific-pathogen free facility at UT MD Anderson Cancer Center(MDACC) in Houston, Texas and were maintained in an Associationfor Assessment and Accreditation of Laboratory Animal Care (AAA-LAC) approved facility, and in accordance with current regulationsof the United States Department of Agriculture and Department ofHealth and Human Services. Mice were 3–4 months of age at thestart of the studies and were housed 5–8 per cage, exposed to12-h light dark cycles, and given free access to sterilized pelletedfood (Prolab Animal Diet, Purina Mills Inc., St. Louis, MO) and ster-ile acidified water. The experimental protocol was approved by,and in accordance with, institutional guidelines established bythe MDACC Institutional Animal Care and Use Committee.

2.2.2. IrradiationGroups of 5 or 8 un-anesthetized mice were loosely restrained

in a 15 l � 15 w � 2 h cm well ventilated Lucite box and exposedto a single dose of whole body irradiation (WBI) ranging from 6.5to 12 Gy using 300 kVp X-rays at a dose rate of 1.84 Gy/min + 0.045 min.

2.2.3. Test agentsWR 2721 and its polymer complex were synthesized at the

Institute of Polymers, Bulgarian Academy of Sciences then sent toUT MD Anderson Cancer Center for animal studies. The polymercomplex and WR 2721 were dissolved in saline on a shaker for30 min to 1 h and stored at 4 �C for the duration of each experi-ment. Treatments were administered by ip injection at a dose of50 or 100 mg/kg in a volume of 0.5 ml per mouse 30 min prior toWBI.

2.2.4. Jejunal microcolony stem-cell assayThe in vivo intestinal microcolony assay (Mason et al., 1989a;

Withers and Elkind, 1970) was used to quantify the survival of

POEClP

POEHP

CH3O-P-O(CH2CH2O) P-O(CH2CH2O)_

O O

Cl Cl (n-1

1313

CH3O-P-O(CH2CH2O) P-O(CH2CH2O) P __OO O

H HH (n-1)

1313

nH2O, Et3Nr.t.

PHOEP

CH3O-P-O(CH2CH2O) P-O(CH2CH2O) P __OO O

OH OHOH(n-1)

1313

Scheme 1. Synthesis of poly(oxyethylene chlorophosphate) P

jejunal crypts 3 days 14 h following WBI. Mice were euthanizedby carbon dioxide inhalation and a 2-cm segment of jejunumwas excised and fixed in 10% neutral-buffered formalin prior toroutine processing for histology. Four transverse sections permouse were cut at a thickness of 4 lm. Slides stained with hema-toxylin and eosin (H&E) were scored at 100�magnification for sur-viving crypts per circumference of jejunum. Only those cryptscontaining at least 10 viable cells were scored. The number of sur-viving crypts was converted to number of surviving stem cells percircumference of jejunum using Poisson statistics to correct formultiple stem cell survival per crypt. Data are represented asmeans ± SEMs. P values were determined using Student’s t testand differences considered significant at P values of 0.05 or less.

2.2.5. LD50/30 assayMice were exposed to single doses of WBI ranging from 6.5 to

11.25 Gy. Mice were observed twice daily following WBI for signsof treatment related morbidity. When mice became moribund(exhibited hunched posture, ruffled fur, persistent diarrhea,

N

N

N

O

OO

Cl Cl

Cl

N

N

N

O

OO

H H

H

n+

n

TCIA

r.t.

_

ICA

P OH_O

Cl)

OH

OH

OEClP and poly(hydroxyoxyethylene phosphate) PHOEP.

Table 1Jejunal crypt cell survaival.

Treatment group Crypts/circumference jejunum mean ± SE

Control (no treatment) 160.5 ± 3.9012 Gy WBI only 21.2 ± 2.78WR 2721 100 mg/kg + 12 Gy WBI 38.6 ± 2.55*

Polymer complex of WR 2721 100 mg/kg + 12 Gy WBI 55.1 ± 6.33*,+

* Significant difference Vs 12 Gy WBI only (P < 0.05).+ Significant difference Vs WR 2721 100 mg/kg + 12 Gy WBI only (P < 0.05).

Fig. 1. Overall survival of mice treated with 9 Gy whole body irradiation. TheKaplan–Meier plot shows percent survival plotted as a function of days afterirradiation. Three groups of 10 mice each were exposed to 9 Gy WBI alone (blackcircles), WR2721 ip at a dose of 50 mg/kg 30 min prior to WBI (blue circles), or thepolymer complex of WR2721 at an ip dose of 50 mg/kg 30 min prior to WBI (redcircles). (For interpretation of the references to color in this figure legend, the readeris referred to the web version of this article.)

12 N. Koseva et al. / European Journal of Pharmaceutical Sciences 65 (2014) 9–14

labored breathing or more that 20% weight loss), they were eutha-nized by CO2 inhalation. Latency to time of euthanasia and percentlethality was determined on the 30th day following WBI (Masonet al., 1989a,b; Yuhas and Storer, 1996). Radiation dose–responsecurves for lethality were constructed and fitted using logit analysisand the radiation dose resulting in 50% lethality by day 30 (LD50)was calculated with 95% confidence limits for each treatmentgroup. When a range of radiation doses was used to constructthe full radiation dose response curve, P values were calculatedusing the likelihood ratio test. When only one radiation dose wasused to assess latency, P values were calculated using the Wilco-xon–Breslow–Gehan test of equality of survivor functions. Differ-ences were considered significant at P values of 0.05 or less.

Fig. 2. Radiation dose survival curves 30 days following treatment with radiationalone (open circles and dashed black line), WR2721 100 mg/kg 30 min before WBI(black squares and solid black line), or WR2721 polymer complex 100 mg/kg30 min before WBI (red triangles and solid red line). The degree to which WR2721or the polymer complex of WR2721 modulates the incidence of the fatal radiationinduced bone marrow syndrome was compared at the 50% response level (LD50).Bars represent 95% confidence limits at the LD50 level. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version ofthis article.)

3. Results and discussion

3.1. Synthesis of the polymer carrier

POEHP based on PEG with nominal molecular weight of 600 Dawas synthesized via the conventional two-stage polycondensationprocess (see Scheme S1). A detailed analysis of the NMR spectraldata for elucidation of the polymer structure is presented in theSupplementary data. Based on the 1H and 31P{H} NMR data thedegree of polymerization of POEHP was 19, i.e. Mn � 12,000. SECmeasurements showed that the molecular weight distribution ofPOEHP (Mw/Mn) was 1.38.

POEHP was used as precursor polymer for the preparation ofpoly(hydroxyoxyethylene phosphate) (PHOEP) that included twosteps. Firstly, poly(oxyethylene chlorophosphate) (POEClP) was

synthesized by oxidation of POEHP with trichloroisocyanuric acid(TCIA) (Troev et al., 2012) (Scheme 1). Addition of equimolaramount of water to POEClP furnished poly(hydroxyoxyethylenephosphate) (PHOEP) in high yield, which is confirmed by 1H,13C{H} and 31P{H} NMR spectra of the product (Figs. S1A, S1B,S1C). The 31P{H} NMR spectrum of PHOEP displayed signals at7.62 (1.35%), 2.17 ppm (3.55%) and 0.90 ppm (95.10%) (Fig. S1C).The signals at 2.17 and 0.90 ppm in the 31P NMR spectrumhave been assigned to the phosphorus atoms in the endgroups CH3OAP(O)(OH)OCH2A and in the repeating units ACH2OP(OH)(O)OCH2A, respectively. The signal at 7.62 ppm appears in31P NMR spectrum as a doublet of triplets with 1J(P.H) = 653.12 Hzand can be assigned to a phosphorus atom bonded to a hydrogenatom in the end group HOP(H)(O)OCH2A, i.e. a small fraction ofthe PAH groups (1.35%) remained unconverted into PAOH.

3.2. Synthesis of the polymer complex of WR 2721

The interaction of amifostine with PHOEP lead to the forma-tion of a salt structure (Scheme 2). The 1H, 13C{H}, 31P{H} NMRspectra of the complex are presented in the supplementary mate-rial (Figs. S2A, S2B, S2C). The formation of ionic bond betweenWR 2721 and poly(hydroxyoxyethylene phosphate) is confirmedby the considerable shift in the signal for the phosphate unitsin the polymer (see Figs. S1C and S2C). In the 31P{H} NMR spec-trum of the complex (Fig. S2C) the main signal for the phosphatestructure of the polymer appears at 0.66 ppm, i.e. the signal isshifted to the high field compared to the signal for the

Table 230 Day mortality following whole body irradiation.

Treatment group LD50a 95% CIb PFc

Radiation only 7.845 (7.596–8.091)WR 2721 100 mg/kg + Radiation 10.099 (9.824–10.404) 1.29*

Polymer complex of WR 2721 100 mg/kg + Radiation 10.561 (10.259–10.926) 1.35# +

a LD50 = Radiation dose resulting in 50% lethality within 30 days.b CI = confidence interval.c PF = protection factor is equal to the LD50 of drug + radiation divided by LD50 of radiation only.* Significant difference vs. radiation only (P = 0.0000000000008).# Significant difference vs. radiation only (P = 0.000000000001).+ Significant difference vs. WR 2721 + radiation (P = 0.049).

N. Koseva et al. / European Journal of Pharmaceutical Sciences 65 (2014) 9–14 13

phosphorus atom in poly(hydroxyoxyethylene phosphate)(0.90 ppm) due to the presence of the negatively charged oxygenatom bonded to the phosphorus (Troev and Roundhill, 1988).Additional evidence is found in the FT-IR spectrum of the poly-mer complex of WR 2721 – the P@O band is shifted from1259 cm�1 in the PHOEP spectrum to 1243 cm�1 in that of thecomplex (Troev and Borissov, 1983).

3.3. Biological testing

Initial studies comparing the radioprotective effect of WR2721to that of the polymer complex of WR2721 utilized the jejunalmicrocolony assay. A single 100 mg/kg dose of WR2721 or thepolymer complex of WR2721 were given 30 min prior to a singledose of 12 Gy WBI. Surviving crypts per jejunal cross section werescored histologically from tissue sections collected 3 days and 14 hafter irradiation, and the number of surviving crypt stem cells wascalculated using Poisson statistics.

The results showed (Table 1) that both agents had a radiopro-tective effect on crypt cell survival. However, the polymer complexof WR2721 significantly increased mean crypt cell survival abovethat of WR2721 from 38.6 crypt cells to 55.1 (P < 0.05). The protec-tion factor for the polymer complex relative to radiation alone was2.6.

Radiation dose response studies were then performed toassess the degree to which the polymer complex of WR2721could modulate the radiation induced bone marrow syndrome(Mason et al., 1989a,b). Latency in days between WBI exposureand lethality and incidence of lethality within 30 days was com-pared to that of WR2721 and the polymer complex of WR2721.The initial study used 9 Gy radiation alone or 50 mg/kg of eachagent 30 min before the same dose of radiation. Time to deathin days for each animal following irradiation was recorded asthe latency. Regardless of treatment, all mice succumbed to thefatal bone marrow syndrome within 3 weeks of radiation expo-sure. Fig. 1 shows there was no difference in latency betweenradiation only and the group treated with the sub-optimal dose(Yuhas, 1971) of WR2721. However, those mice treated withthe polymer complex of WR2721 lived marginally significantlylonger (P 0.0514) than those treated by WBI alone. Subsequentstudies used a higher dose of WR2721 (100 mg/kg) which wasexpected to have a more robust protective effect on radiationinduced bone marrow lethality based on previous studies(Yuhas, 1971). Complete radiation dose survival curves were con-structed using a range of radiation doses between 6.5 and11.25 Gy. Fig. 2 shows the radiation response curves for LD50in mice exposed to radiation alone, or in mice that received100 mg/kg WR2721 or the polymer complex of WR2721.

Both agents showed a significant radioprotective effect(P < 0.000000000001) (Table 2), however, pre-irradiation treat-ment with the polymer complex of WR2721 resulted in a signifi-cantly greater protection factor of 1.35 compared to that forWR2721, 1.29 (P 0.049) at the LD50 level.

4. Conclusion

The immobilization of WR 2721 – a cysteamine analog on poly-phosphoester via ionic bonds leads to the formation of a new poly-mer complex of the radioprotector. The influence of polymercarrier on the radioprotective efficacy of WR 2721 was evaluatedin experiments on mice irradiated with gamma rays. Radiationprotection factors were assessed for WR2721 and for the polymercomplex. In all cases, the polymer complex was a significantly bet-ter radiation protector than the parent compound. Protection fac-tors for the polymer complex ranged from 2.6 for intestinal stemcell survival to 1.35 for 30 day survival (LD50) following wholebody radiation exposure.

Acknowledgment

Financial support of this work was provided by the National Sci-ence Fund of Bulgaria (National Center for Advanced Materials(UNION) Module 2 ‘‘New materials in medicine and pharmacy’’Contract DCVP 02-2/2009.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ejps.2014.08.006.

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