Extended release of a novel antidepressant, venlafaxine,based on anionic polyamidoamine dendrimers andpoly(ethylene glycol)-containing semi-interpenetratingnetworks

Hu Yang, Stephanie T. LopinaDepartments of Chemical Engineering and Biomedical Engineering, 302A Whitby Hall, The University of Akron,Akron, Ohio 44325-3906

Received 15 June 2004; revised 25 August 2004; accepted 7 September 2004Published online 12 November 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30220

Abstract: The multiple daily administration of venlafaxine,a novel third-generation antidepressant, was reduced basedon polyamidoamine and polyethylene glycol (PEG)-contain-ing semi-interpenetrating network (IPN), respectively. Ven-lafaxine was covalently linked to water-soluble G2.5 anionicpolyamidoamine dendrimer via a hydrolyzable ester bond.Semi-IPN hydrogels were prepared by crosslinking acryl-amide in the presence of PEG, and venlafaxine with prede-termined amounts was loaded in situ. Dendrimer–venlafax-ine conjugate and semi-IPNs were characterized by proton

nuclear magnetic resonance and Fourier transform infrared,respectively. The effect of PEG concentration and molecularweight was studied and discussed for an optimal controlledrelease. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res72A: 107–114, 2005

Key words: venlafaxine; Starburst� polyamidoamine(PAMAM) dendrimer; polyacrylamide hydrogel; polyethyl-ene glycol; semi-interpenetrating network (IPN); extendedrelease

INTRODUCTION

Venlafaxine (1-[2-(dimethylamino)-1-(4-methoxy-phenyl)-ethyl] cyclohexanol hydrochloride) is a novelthird-generation antidepressant having a bicyclicstructure different from tricyclic, tetracyclic, or othercommercially available antidepressants.1 Venlafaxineand its active metabolite, O-desmethylvenlafaxine(ODV) (Fig. 1) were proved to have improved efficacy,faster speed of onset of effect, and greater safety in thetreatment of depression, generalized anxiety disorder,and social anxiety disorder in adults compared withprevious medicines, such as the selective serotoninreuptake inhibitors.2,3 In addition, venlafaxine workseffectively against a broad range of depressive condi-tions, both mild-to-moderate and severe, whether oc-curring in in-patients or out-patients.4 However, a

short steady-state elimination half-life (3–4 h for ven-lafaxine and 10 h for ODV5,6; both are independent ofdose) makes it requisite to have a multiple daily ad-ministration in order to keep a stable therapeuticlevel.7,8 Both the immediate- and extended-release for-mulations have demonstrated efficacy in reducingsymptoms of depression. However, the extended-re-lease formulation has advantages in increasing patientcompliance and convenience as well as a superiorbenefit/risk ratio to venlafaxine immediate re-lease.9–11 Aside from the marketed formulation andrecent work by Makhija and Vavia,12 in this article,two new strategies are presented based on poly-amidoamine (PAMAM) dendrimers and polyethyleneglycol (PEG)-containing semi-interpenetrating net-work (IPN) hydrogels, and the extended release ofvenlafaxine was investigated.

Dendrimers are synthetic globular three-dimen-sional structures with very low polydispersity andhigh functionality, which were first synthesized in theearly 1980s.13–15 Compared with linear polymers, theirmany accessible reactive end groups at the surface aremore suitable to combine agents of interest throughproper chemical modification, and thus achieve ahigher drug loading within a compact scaffold.16 Theirinside cores have been proved feasible for encapsula-

Correspondence to: S. T. Lopina, e-mail: lopina@uakron.eduContract grant sponsor: National Science Foundation CA-

REER award; contract grant number: BES-9984840Contract grant sponsor: University of Akron Faculty Re-

search Grant; contract grant number: FRG-1484Contract grant sponsor: Sigma Xi Grant-in-Aid of Re-

search

© 2004 Wiley Periodicals, Inc.

tion of guest molecules as well.17–20 Therefore, theyhave been increasingly attracting attention for drugdelivery or gene therapy.15,21–30 In this study, ven-lafaxine was covalently linked to water-soluble G2.5anionic PAMAM dendrimer (Fig. 2) via a hydrolyz-able ester bond. A high drug loading could beachieved. The bioavailability of such conjugated drugwas proved by hydrolysis studies. A second strategyis to use PEG-containing polyacrylamide (PAAm) hy-drogels. The low mechanical strength of PAAm hy-drogels was improved by incorporating a linear poly-mer in the hydrogels. Semi-IPN hydrogels wereprepared by crosslinking acrylamide (AAm) in thepresence of PEG, and venlafaxine with predeterminedamounts was loaded in situ. The effect of PEG on the

network structural properties and drug release ratewas studied. The strategies we present here could bereferred to a general procedure for drugs demandingextended release to reduce daily administrations.

MATERIALS AND METHODS

Materials

Starbust� PAMAM dendrimer generation 2.5 (10 wt %solution in methyl alcohol, containing 32 surface carboxylategroups, fw 6267), PEG (Mn � 1000; 8000), p-nitrophenylchloroformate, triethylamine, dimethylformamide (DMF),AAm, N,N�-methylene bisacrylamide (MBA), potassiumpersulfate, dicyclohexyl-carbodiimide (DCC), 4-dimethyl-aminopyridine (DMAP), and phosphate-buffered saline(PBS, 10�) were purchased from Sigma-Aldrich (St. Louis,MO). Venlafaxine hydrochloride (simply referred to as ven-lafaxine) was received as a gift sample from Wyeth-AyerstResearch (Monmouth Junction, NJ). Fresh distilled waterwas used in the experiments unless specified.

Spectroscopic characterization

Proton nuclear magnetic resonance (1H NMR) spectrawere recorded on a Varian Gemini 300-MHz spectrometer.

Figure 1. Chemical structures of venlafaxine and its activemetabolite, ODV.

Figure 2. Schematic structure of G2.5 PAMAM dendrimer (generally, commercial carboxylate dendrimer is in the carbox-ylate sodium salt form which is stored in methanol).

108 YANG AND LOPINA

The internal standard was tetramethylsilane. Deuterium ox-ide (D2O, 99.9%) was obtained from Cambridge IsotopeLaboratories. Each sample was dissolved in deuterated sol-vent, filtered, and degassed before measurement. The chem-ical shift of D2O is 4.8 ppm. Fourier transform infrared(FTIR) spectra of completely dehydrated thin hydrogel filmswere obtained on a Nicolet Nexus 670 spectrometer. Ultra-violet–visible (UV–vis) absorbance data for venlafaxine at 274nm were gained on a Beckman DU640 spectrophotometer.

Preparation of venlafaxine–PAMAM conjugates

One hundred twenty milligrams of G2.5 PAMAM (19.2�mol) was acidified by 5N HCl to pH 3.0, then was dried invacuum and redissolved in 10 mL of DMF according to aprevious report.31 Venlafaxine was coupled to the carboxylgroups of dendrimers following the procedures providedpreviously.32,33 In brief, 288.8 mg of venlafaxine (920 �mol)was dissolved in DMF (2 mL). To the solution were added112 mg of DMAP (920 �mol) and 190 mg of DCC (920 �mol).The mixture was stirred for 12 h at room temperature. Salt (i.e.,DCU), formed in the reaction, was removed by filtration. Thesolvent was removed under reduced pressure using a rotaryevaporator. The product was purified by using dialysis wheredistilled water was refreshed until no detectable absorbancewas observed in newly refreshed water by a UV–vis spectro-photometer. 1H NMR spectrum (Fig. 3) was used to confirmthe conjugate structure and roughly estimate the average num-ber of venlafaxine molecules per a dendrimer.

1H NMR spectrum of venlafaxine–PAMAM conjugate(300 MHz, D2O): � 1.01–1.78 ppm (10n, m, methylene incyclohexane of venlafaxine), 2.60–2.67 ppm (m, protons ofdendrimer), 2.75–2.88 ppm (6n, s, methyl connecting N ofvenlafaxine), 3.05–3.16 ppm (1n, dd, methine of venlafaxine),3.18–3.21 ppm (m, protons of dendrimer), 3.32–3.44 ppm (m,

protons of dendrimer), 3.56–3.77 ppm (2n, m, methyleneconnecting N of venlafaxine), 3.68–3.72 ppm (m, protons ofdendrimer), 3.83–3.86 ppm (3n, s, methyl connecting O ofvenlafaxine), 7.02–7.09 ppm (2n, d, aromatic protons of ven-lafaxine), 7.35–7.41 ppm (2n, d, aromatic protons of ven-lafaxine), here n represents the average number of venlafax-ine molecules per dendrimer.

Preparation of semi-IPN hydrogels and swellingmeasurements

Semi-IPN hydrogels were prepared by the aqueous free-radical polymerization of AAm at the presence of PEG.34,35

A series of PEG-containing (Mn � 1000 or 8000) PAAmhydrogels was prepared by varying composition of PEG (23,42, and 65 wt %) in the hydrogels. Typically, the synthesis of65 wt % PEG 1000-containing semi-IPN was described asfollows: 930 mg PEG (0.93 mM), 500 mg AAm (7.04 mM), 9.9mg MBA (0.064 mM), 10 mg potassium persulfate (0.037mM), and 9.9 g distilled water (0.55M) were mixed with amagnetic stir bar until they were all completely dissolved.After the solution was nitrogen bubbled for 10 min, thereaction was performed in a water bath at 80°C for 30 min.The gel-like mass formed after reaction was then dried at60°C for 18 h. Thereafter, the gel film was air-dried and keptin the dried state before tests.

The following parameters were measured on each sample:wr, the weight of the relaxed hydrogel right after reaction;ws, the weight of the fully swollen gel; and wp, the weight ofthe completely air-dried gel. Based on the above values, thepolymer volume fraction after reaction (relaxed state), �2,r;the polymer fraction after equilibrium swelling (equilibriumsate), �2,s; the number-average molecular weight betweencrosslinks, M� c; and the mesh size, �, were calculated. Theequilibrium swelling degree (qw) was determined as follows:

Figure 3. 1H NMR 300-MHz spectrum of venlafaxine–PAMAM conjugate in D2O.

EXTENDED RELEASE OF VENLAFAXINE 109

qw � ws/wp (1)

In vitro drug release studies

To prove the bioavailability of the conjugated venlafaxine,in vitro hydrolysis of PAMAM-conjugated venlafaxine wasinvestigated. The conjugated drug was incubated in a dial-ysis bag with molecular weight cut-off 3500 in pH 7.4 PBS at37°C. Three milliliters of drug release medium was taken outat each predetermined interval and measured at 274 nm bya UV–vis spectrophotometer. Drug release medium was putback immediately after each measurement.

Before measuring venlafaxine release from hydrogels,venlafaxine-loaded semi-IPNs were prepared. Predeter-mined initial doses of free venlafaxine of 75 and 8 mg,respectively, were loaded in situ when each hydrogel wasprepared by using the procedure presented in the previoussection. Similarly, venlafaxine release from each drug-loaded hydrogel film was measured under the same physi-ological conditions as conjugated venlafaxine. Three millili-ters of fresh PBS was added to the solution afterward.

RESULTS

Synthesis and characterization ofvenlafaxine–PAMAM conjugates

The strategies for the coupling reaction between thecarboxylate surface groups of Starburst� dendrimer andhydroxyl groups are similar to those of small mole-cules.30,36 According to Scheme 1, a direct coupling re-action produces the venlafaxine–PAMAM conjugate us-ing the condensation reagent DCC and the catalystDMAP. Venlafaxine was covalently connected to thedendrimer core through a hydrolytic ester bond. G2.5PAMAM with approved low toxicity and desired bio-logical properties was chosen as a model anionicPAMAM dendrimer. An ideal structure would have adendritic core with a venlafaxine molecule at the end ofeach branch. 1H NMR was used to measure the drugloading. Although 1H NMR could not detect structuralfragment errors like matrix-assisted laser desorptionionization–time-of-flight mass spectrometry (MALDI-TOF MS), it is still a useful method to estimate thepercentage of dendrimer surface occupation by drugmolecules, and the results were comparable to thoseobtained from MALDI-TOF MS.37 The 1H NMR spec-

trum (Fig. 3) proved that the proton peaks from conju-gated venlafaxine are identical to those in unmodifiedvenlafaxine. The multiple proton peaks from the den-drimer are found between 2.60 and 3.72 ppm. Some ofthe proton peaks from the dendrimer core could not bedifferentiated from those of drug around the same range.Therefore, the proton intensity of dendrimer was calcu-lated by subtracting the proton intensity of venlafaxineproportionally by referring to the intensity of the aro-matic protons of venlafaxine between 7.02–7.41 ppm.According to the rough estimation, nearly 100% of thedendrimer surface sites were coupled to venlafaxinemolecules. This also suggests that the venlafaxine mole-cule is small enough to avoid steric hindrance whichotherwise would lead to an incomplete coupling reactionwith functional groups at the dendrimer surface.

Synthesis and characterization of PEG-containingsemi-IPNs

The amount of crosslinker, MBA, determines thedegradation of the MBA-crosslinked hydrogels. Hy-drogels with 0.3–0.6% crosslinker degraded over 2–10days. At concentrations �1%, the hydrogels were con-sidered not to degrade within the observation timescale.38 In this study, nondegradable PAAm hydrogelswere synthesized. The effect of uncrosslinked PEG onthe network structure was studied by varying PEG con-tent in concentration and molecular weight. To minimizethe effect of other components on the network structuralproperties, concentrations of monomer, crosslinker, andcatalyst were maintained constant.

The chemical components of semi-IPN hydrogelswere analyzed using an FTIR spectrum (e.g., Fig. 4 forsemi-IPN1). The spectrum shows all characteristicbands of crosslinked AAm. A broad band represent-ing NOH stretching in the 3100–3600 cm1 region isfound from the PAAm network. Other characteristicbands appearing in the spectrum for PAAm are foundin the 1650–1700 cm1 region due to CAO stretching.The combined bands of NOH deformation and CONstretching occur in the 1580–1650 cm1 region. Thepeak near 1450 cm1 is due to COH deformation andthe broad peak below 1000 cm1 is due to NOHout-of-plane bending. Although most of the character-istic bands of PEG are eclipsed by PAAm, a strongband at the 2800–3000 cm1 region due to COHstretching and a new band at 1080–1160 cm1 due toCOO stretching could be identified from PEG andproved the presence of PEG in the semi-IPN.

The effect of PEG on swelling behaviors andnetwork structural properties

Swelling behavior of synthesized networks was in-vestigated. The swelling ratios of investigated semi-

Scheme 1. Synthesis of venlafaxine–PAMAM conjugate.

110 YANG AND LOPINA

IPNs are listed in Table I where PEG concentrationswere varied between 23–65 wt %. As seen in Table I,for PEG 1000-containing semi-IPNs, when PEG con-tent increases from 23% to 65 wt %, the hydrogelequilibrium swelling ratio decreases from 16.8 to 9.9.For both 65 wt % PEG 1000- and PEG 8000-containingsemi-IPNs, their swelling ratios are much lower than23 wt % and 42 wt % PEG-containing semi-IPNs. The65 wt % PEG 8000-containing semi-IPNs have thelowest equilibrium swelling ratio among all synthe-sized semi-IPNs. After the first swelling, the swollenhydrogels were completely dried and then swollenagain. The second equilibrium swelling ratios at 25°and 37°C are also listed in Table I. Among them,semi-IPN6, that is, 65 wt % PEG 8000-containing semi-IPN, has the lowest first and second swelling ratios.

A correlation between the mesh size and the polymer

volume fractions in the swollen state was establishedaccording to the work done by Canal and Peppas.39 Themesh size was calculated using the following equation:

� � �2,s1/3l2M� cCn/Mr�

1/2 (2)

where l (1.54 Å) is the carbon–carbon bond length, Mr(71.08 for PAAm) is the molecular weight of the repeat-ing unit, and Cn (8.5 for PAAm40) is the polymer char-acteristic ratio. The molecular weight between junctions,M� c, was calculated according to the Flory equation.41

1

Mc

� �ln�1 � �2,s � �2,s � ��2,s

2

�V1�2,r���2,s

�2,r�1/3

�12 ��2,s

�2,r�� (3)

Herein, � is the density of dry PAAm gel (using 1.27 g/mLas reported42), V1 is the molar volume of the swelling

Figure 4. FTIR spectrum of PAAm–PEG semi-IPN1.

TABLE IComposition and Swelling Measurements of PAAm–PEG Semi-IPNs

Semi-IPNNo.

Feed Composition (wt %) Equilibrium Swelling Ratio

AAm PEGM� n ofPEG

First Swelling Second Swelling

25°C 25°C 37°C

1 77 23 1000 16.8 � 0.6 17.1 � 0.3 22.1 � 0.82 58 42 1000 9.9 � 0.9 13.3 � 0.8 17.0 � 1.53 35 65 1000 9.9 � 0.1 13.5 � 1.5 16.8 � 2.54 77 23 8000 13.1 � 0.4 12.9 � 0.7 15.8 � 0.75 58 42 8000 15.0 � 0.7 16.3 � 0.2 20.7 � 0.56 35 65 8000 7.3 � 0.4 6.2 � 0.2 6.8 � 0.2

EXTENDED RELEASE OF VENLAFAXINE 111

agent (18.0 mL/mol for water), and � is the Flory poly-mer–solvent interaction parameter (0.48 for PAAm gel–water system35). In Equations (2) and (3), the polymervolume fractions of the gel in the relaxed state (�2,r) andthe swollen state (�2,s) can be determined as follows:

�2,r �wp

wp � �wr � wp ��

�s� (4)

�2,s �wp

wp � �ws � wp ��

�s� (5)

Here, �s is the solvent density (i.e., 1.0 g/mL for water).As to our samples, �2,s varies between 0.028 and

0.053. Based on De Gennes’s theory,43,44 when 0.01 ��2,s � 0.1, the correlation between � and �2,s follows:

� � �2,s0.50 (6)

The data obtained from the first swelling measure-ment were analyzed using Equation (6) with a linearregression. The result (Fig. 5) indicates that the follow-ing parameters in Equation (7) give a good correlationcoefficient as r2 � 0.9755.

� � � 11.241 � 1.5505�2,s0.50 (7)

According to the first swelling measurement of thehydrogels, the mesh size (�) ranges from 18 to 45 nmfor PEG 1000- and 8000-containing semi-IPNs whenPEG concentrations vary between 23 and 65 wt %.

In vitro release studies

The in vitro release study was performed in pH 7.4PBS medium at 37°C. The amount of drug released

was measured by a UV–vis spectrophotometer at pre-determined time intervals. Hydrolysis study of ven-lafaxine–dendrimer conjugate indicates that the drugwas released in a sustained way, that is, only half ofthe conjugated drug was released within 18 h (Fig. 6).

Sixty-five percent PEG 8000-containing semi-IPNwas chosen as a drug loading matrix because it has thelowest swell ratio and good stability under reswellingcondition. As seen in Figure 7, for 75 mg venlafaxine-loaded matrix, 20% of the venlafaxine molecules werereleased within 15 min from the matrix because of theburst effect. Ninety-two percent of the venlafaxinewas released within 6 h. The drug release data were fitto a first-order kinetic equation. In contrast, for 8 mgvenlafaxine-loaded matrix, the drug release rate wassignificantly reduced. A time-independent zero-orderrelease was obtained. The burst release was alsoavoided. Only 50% of venlafaxine was released from thematrix within 6 h. Drug loading has a pronounced im-

Figure 5. Correlation between the mesh size (�) and theequilibrium polymer volume fraction (�2,s) for all semi-IPNsprepared in this study.

Figure 6. In vitro hydrolysis of venlafaxine–PAMAM con-jugates in PBS at 37°C.

Figure 7. Cumulative release of venlafaxine from semi-IPN6 in PBS at 37°C.

112 YANG AND LOPINA

pact on the rate of drug release. A larger loading cancause a faster movement of the solvent front as well asfacilitate relaxation of the networks of the gel leading toa higher swelling ratio. It is believed that these are majorreasons why a lower drug loading is preferable in orderto achieve a slower drug release. Similar results werefound in other hydrogel loading systems.45

DISCUSSION

Starburst� PAMAM dendrimers with a series ofgenerations is one of most successfully commercial-ized dendrimers which was developed by Tomalia etal.13,14 (Dendritech Inc., Midland, MI). PAMAM den-drimers are called artificial proteins because of theirsize and contour similar to many important naturalglobular proteins.22 They are more stable than proteinswhen they are subject to pH, temperature, light, or otherdenaturing conditions. Such unique structure propertiesmake them good replacements of natural proteins indrug delivery and gene therapy as shown by biologicalstudies.22,26 Their toxicity was found to be dose- andgeneration-dependent in vitro. Low generations (G5.0 orlower) are not considered toxic. Moreover, there is noevidence of immunogenicity for tested PAMAM den-drimers.46 Anionic dendrimers were neither lytic norcytotoxic in a broad concentration range.28

Although pure PAAm hydrogels show rather highswelling ratio, in a highly swollen state, their mechan-ical frangibility limits their applications for drug de-livery. It was found that the addition of a secondcomponent would increase the mechanical strength ofthe original hydrogel.47 It was also found that a fur-ther increase in the concentration of PEG could in-crease the swelling ratio, however, at the cost of asignificant loss of its mechanical strength. This mayoccur because when more PEG molecules occupy thespace among monomers, the integrity of the wholenetwork is disrupted, thus resulting in the loss ofmechanical strength. The migration of PEG moleculesout of the network during the first swelling allowsmore water to diffuse into the matrix during the sec-ond swelling, thus causing the second swelling ratio tobe higher. As seen in Table I, higher molecule weightor higher content of PEG could increase the degree ofentanglement or density, thus enhancing pseudo-crosslinks in the network, which could delay watermolecule penetration.34 Two characteristic parame-ters, that is, mesh size and equilibrium swelling de-gree, were influenced by PEG molecular weight, con-centration, and hydrogel swelling history. The drugrelease rate was affected by the swelling behavior andmesh size. The amount of drug entrapped in the hy-drogel influence the drug release rate. The higher the

drug loading, the greater the burst and the faster therelease rate.

Here, a new drug delivery strategy is proposed byloading dendrimer-based venlafaxine carriers into hy-drogels. The diffusion rate of the relatively large den-drimeric scaffold from the hydrogel is assumed to bemuch slower than the diffusion of unconjugated drug.This may provide a benefit for this dosage form if it isapplied to local drug therapy. For example, if thedrug-loaded network is exposed to plasma, it willencounter such enzymes as carboxylesterase. The en-zyme cleaves the ester bond more quickly than acidsdo. Exposure to such an enzyme might cause therelease rate to be faster than expected and less con-trollable. In this case, it is expected that the networkcould help retard any possible invasion of enzyme orother biomacromolecules by confining dendrimer con-jugates to the inner pores or at least by significantlyretarding the diffusion. In this way, hydrolysis occur-ring within the network would be solely subject to theattack of water and acid in vivo. Further study will beneeded to correlate the mesh size to the diffusion ofthe dendrimer–drug conjugate.

CONCLUSIONS

Conjugation of venlafaxine to PAMAM dendrimerscould significantly extend the release of venlafaxine,controlled by hydrolysis. Further extension and con-trol of release rates are achieved by incorporating theconjugated dendrimers into PEG-containing semi-in-terpenetrating polymer networks. The release rate iscontrolled by optimizing PEG concentration and mo-lecular weight.

The authors gratefully acknowledged the receipt of ven-lafaxine as a gift sample from Wyeth-Ayerst Research (Mon-mouth Junction, NJ).

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