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`
`Topical disposition of two strengths of a I-125-rhEGF jelly in rat skin
`wounds
`
`Article  in  Biopharmaceutics & Drug Disposition · July 2004
`
`DOI: 10.1002/bdd.393 · Source: PubMed
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`

`

`BIOPHARMACEUTICS & DRUG DISPOSITION
`Biopharm. Drug Dispos. 25: 193–201 (2004)
`Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bdd.393
`
`Topical Disposition of Two Strengths of a 125I-rhEGF Jelly in
`Rat Skin Wounds
`
`J. Ducongea,*, P.A. Pratsb, C. Valenzuelab, A. Aguilerab, I. Rojasb, M.A. Becquera, D. Alvareza, L. Estradac,
`S. Alfonso-Ort´ııza,y, E. Hardy-Randob, O. Garc´ııa-Pulpeirod and E. Ferna´ndez-Sa´ncheza
`a Laboratory of Biopharmaceutics, Department of Pharmacology, Institute of Pharmacy and Foods, University of Havana, Ave 23 and 222,
`La Coronela, Havana 36, CP 13600, Cuba
`b Center for Genetic Engineering and Biotechnology, Havana, Ave 31 e/ 158 y 190. P.O. Box 6162, Havana 10600, Cuba
`c Department of Pharmacy, ‘C.J. Finlay’ University Hospital, Havana 10600, Cuba
`d Pharmaceutical Laboratories ‘Roberto Escudero’, Research and Development Section, Havana, Cuba
`
`ABSTRACT: Growth factors have proved to be an effective therapeutic strategy. However, some
`controversies have arisen concerning their efficacy in topical wound treatments. Stabilization of
`epidermal growth factors at the wound site and long-lasting receptor occupancy are important
`factors for wound repair. This study evaluated the cumulative profiles of two jellies containing 10 or
`20 mg of 125I-rhEGF per gram of jelly, in a rat full-thickness skin lesion model. The prolonged time-
`courses at the wound sites for both strengths compared with saline solutions previously evaluated
`using a similar skin lesion model are reported. It seems that these two topical formulations that
`provide more sustained amounts of 125I-rhEGF over the period of sampling, would probably
`achieve the required wound healing response in terms of cell proliferation, collagen deposition and
`protein synthesis. Further studies need to be developed in order to elucidate whether such an in vivo
`disposition pattern is consistent with an earlier and stronger promotion of wound healing
`events. Copyright # 2004 John Wiley & Sons, Ltd.
`Key words: 125I-rhEGF; jelly; topical disposition; strength; non-linearity
`
`Introduction
`
`The promotion of dermal and epidermal regen-
`eration is a crucial aim in the treatment of acute
`and chronic wounds. Growth factors have
`proved to be an effective therapeutic strategy.
`However, controversy exists over the efficacy of
`many topical wound treatments, particularly
`growth factors, and questions have arisen as to
`the real value of these agents.
`
`*Correspondence to: Laboratory of Biopharmaceutics, Depart-
`ment of Pharmacology,
`Institute of Pharmacy and Foods,
`University of Havana, Ave. 23 and 222, La Coronela, Havana
`36, CP 13600, Cuba. E-mail: jduconge@yahoo.com.mx
`y Current address: Center of Immunoassay, SUM 1, Ave 25 and
`134, Playa, Havana, Cuba.
`
`For instance, exogenous epidermal growth
`factor (EGF) accelerates wound healing, but
`treatment effects are often modest.
`It
`is the
`opinion of some researchers that failures are
`due to the existence of local factors such as
`proteases in the wound fluid that make local
`protein administration ineffective [1, 2, 3]. It is
`clear that new delivery systems and therapeutic
`strategies need to be developed to improve
`dermal and epidermal regeneration.
`Several findings indicate that stabilizing EGF
`at the wound site and achieving long lasting
`receptor occupancies are important factors in
`permitting the expression of its healing effects
`and suggest that topical formulations stabilizing
`EGF would be suitable dosage forms for the
`acceleration of wound repair.
`
`Copyright # 2004 John Wiley & Sons, Ltd.
`
`Received 29 January 2003
`Revised 20 October 2003
`Accepted 26 October 2003
`
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`194
`
`J. DUCONGE ET AL.
`
`Our research group has been working with a
`silver sulfadiazine-based rhEGF cream, and has
`obtained contradictory results. It was found that
`silver itself retarded the wound healing process
`besides having deleterious effects on EGF,
`mediated through the generation of silver free
`radicals, although others have found the opposite
`[4].
`Evidence indicates that mitogenic stimulation
`by EGF requires at least an 8–12 h exposure to
`make the majority of the cells divide [5]. Even
`exposures for as short as 2 h have produced a
`significant increase in healing rates, and increas-
`ing the time of exposure further increases the rate
`of wound healing [6].
`Simple and competitive dosage forms are
`adequate for treating skin lesions successfully.
`Our previous report [7] found a rapid disap-
`pearance of 125I-rhEGF solutions from full-thick-
`ness skin wounds in rats and hypothesized that
`other dosage forms such as a jelly or a cream
`could prolong EGF permanence at the lesion site
`without incurring the expense of more compli-
`cated strategies.
`This paper reports prolonged courses of two
`strengths of a 125I-rhEGF jelly at wound sites
`compared with saline solutions previously eval-
`uated in a similar skin lesion model.
`
`Materials and Methods
`
`Growth factor
`
`Highly purified recombinant human EGF was
`produced at the Center for Genetic Engineering
`and Biotechnology (CIGB, Havana, Cuba). Pur-
`ified NaI125 EGF (0.1 mg/ml) radiolabelled by
`the chloramine-T method (Amersham, Arlington
`Heights,
`IL, USA) was used (radiochemical
`purity 93%; efficiency 72% and specific activity
`200 mCi/mg).
`
`Formulation
`The 125I-rhEGF topical
`jelly formulation was
`developed by the Center for Genetic Engineering
`and Biotechnology, Havana, Cuba [8] containing
`125I-rhEGF (0.001%, 10 mg/g, lower strength, or,
`0.002%, 20 mg/g, higher strength), carbopol 940
`
`(0.50%), propylenglycol (10%), NaOH (0.20%),
`(0.010%), methyl-paraben (0.18%),
`EDTA-Na2
`propyl-paraben (0.02%) and sterile water. All
`these aqueous phase excipients were obtained
`from Sigma (St Louis, MO, USA) and Merck
`(Darmstadt, Germany).
`
`125I-rhEGF cumulative profiles in skin lesions
`
`All animal procedures were carried out under the
`approval of the Ethics Committee for Laboratory
`Animal Use at CIGB. All the invasive procedures
`were conducted under sodium pentobarbital
`anaesthesia (40 mg/kg body weight). Adult male
`Sprague-Dawley rats (247–265 g, 7–9 wk old)
`were intraperitoneally anaesthetized and four
`1  1 cm full-thickness skin lesions were made on
`the upper and lower back. Lesions were allowed
`to evolve untreated for 2 h. Then, 400 mg of jelly
`containing either 10 or 20 mg of 125I-rhEGF per
`gram of jelly was delivered to each lesion site.
`Each lesion was considered as an experimental
`unit (n ¼ 12). Three rats per experimental group
`were killed and 12 lesions were sampled at 2, 4, 6,
`12, 24, 48 and 72 h. Skin specimens were excised,
`flushed and then washed twice with ice-cold
`phosphate buffered saline (pH 7.4), and weighed
`before measurement of radioactivity in a 1272
`gamma counter (LKB, Sweden). The resultant
`tissue-associated radioactivity was expressed as
`the percentage of the administered dose per gram
`of tissue from the wound site (%D/g). A 125I-
`rhEGF-radioactivity standard (100% radioactiv-
`ity) was included for correction of radioactivity
`measurements.
`Serial plasma samples were obtained by
`puncture of the retrorbital plexus. Individual
`urine samples were taken by means of sterile
`urine collectors, at the same experimental time
`points. Blood was placed in heparinized tubes
`and centrifuged at 6000 rpm, for 1 min. Finally,
`300 ml aliquots were pipetted into LP3 tubes for
`determining TCA-precipitable plasma-associated
`radioactivity. One hundred microliters of plasma
`was added to the same volume of 30% TCA (v/
`v), vortexed and incubated for 30 min on ice,
`centrifuged for 5 min at 6000 rpm, and the
`sediment
`fraction was counted using a 1272
`gamma counter (LKB, Sweden). The total urine
`volume was registered and 300 ml aliquots were
`
`Copyright # 2004 John Wiley & Sons, Ltd.
`
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`COMPARATIVE 125I-RHEGF JELLY KINETIC PATTERN
`
`195
`
`transferred to LP3 tubes
`excreted radioactivity.
`
`to determine the
`
`Kinetic analysis
`
`Analysis of the drug disposition pooled data was
`performed by a non-compartmental analysis
`(NCA) with a combined linear/log linear trape-
`zoidal rule approach. A time zero value was
`added for extrapolation purposes. The linear
`trapezoidal rule was used up to the peak level,
`after which the logarithmic trapezoidal rule was
`applied. Lambda z is a first-order rate constant
`associated with the terminal (log linear) segment
`of the curve. It was estimated by linear regression
`of the included terminal data points. Goodness of
`fit for the terminal elimination phase, adjusted
`for the number of points used, was also pro-
`vided. The largest adjusted regression was
`selected in order to estimate lambda z, with one
`caveat: if the adjustment did not improve, but
`was within 0.0001 of
`the largest value,
`the
`regression with the larger number of points was
`used.
`Parameters extrapolated to infinity, such as
`AUC (i.e. area under the disposition curve) and
`AUMC (i.e. area under the first moment of the
`disposition curve), were computed based on the
`last predicted level, where the predicted value
`was based on the linear regression performed to
`estimate the terminal
`lambda first-order rate
`constant. Computing these parameters based on
`the last observed level was discouraged in order
`to avoid large estimation errors. The time to peak
`value was determined as the time of the max-
`imum observed level
`(i.e. maximum %D/g)
`considering the entire curve; and the peak level
`was that corresponding to the above mentioned
`time to peak value. For all
`these purposes
`WinNonlin professional software (Version 2.1,
`Pharsight Inc., 1997, NC, USA) was used.
`In the case of flat, modified-release formula-
`tion,
`the plateau time was a more suitable
`parameter than the time to peak. The plateau
`time was defined as the time span of one dosing
`cycle, during which the drug levels deviated
`from the peak level, by less than a clinically
`specified percentage. The time during which 125I-
`rhEGF levels were at least half the peak level
`(50%) was considered by Meier et al.
`[9]
`to
`
`correlate to the width of the efficacy range, and
`corresponded to the half-value duration (HVD).
`For assessing the extent of persistency of the
`modified-release formulation, simple solutions,
`i.e. 125I-rhEGF saline solutions, and modified-
`release jelly formulations, with two 125I-rhEGF
`strengths (10 and 20 mg/g), were chosen under
`the null hypothesis that the peak level would not
`differ by more than 20%. Based on their half-
`value duration ratio (RHVD), the following ratings
`were taken into consideration: RHVD51.5 is no
`retardation; 1.54RHVD53, is weak to medium
`retardation; RHVD53 is strong retardation.
`The 125I-rhEGF in situ availability (Fin situ) from
`the jelly formulation at
`the lesion site was
`determined by the ratio of the AUC for each
`strength to the AUC of the saline solution, using
`the corresponding dose correction factor. For
`these purposes the previously reported experi-
`mental data for saline solutions containing 200
`and 400 ng of 125I-rhEGF were considered [7].
`
`Statistical analysis
`
`tendency and dispersion
`Measures of central
`(mean, median, minimum and maximum values,
`standard deviation and inter-quartile range)
`were calculated in each case. The assumptions
`of normality (Shapiro-Wilks test) and homogene-
`ity of variances (Levene test) among the groups
`were verified at each time. Differences between
`the groups at each time-point were assessed by
`the independent-samples t-test or the non-para-
`metric Mann-Whitney U-test, depending on the
`occurrence of the previously verified assump-
`tions. Also, time variations in every group were
`analysed by the Dunn test. In all cases,
`the
`analysis was made using the software SPSS for
`Windows, version 10.0, and a p value 5 0.008
`was considered statistically significant, using the
`Bonferrony adjustment.
`
`Results
`
`Figure 1 depicts the 125I-rhEGF local disposition
`profiles, after
`topical application of a jelly
`formulation containing two different strengths
`(i.e. 10 and 20 mg/g) in a full-thickness skin lesion
`in rats. Table 1 shows the 125I-rhEGF-
`model
`
`Copyright # 2004 John Wiley & Sons, Ltd.
`
`Biopharm. Drug Dispos. 25: 193–201 (2004)
`
`Apotex, Inc. (IPR2019-00400), Ex. 1018, p. 004
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`

`

`196
`
`J. DUCONGE ET AL.
`
`Figure 1. Disposition profiles after application of a jelly formulation containing each dosage strength under study, i.e. 10 (&) and
`20 mg 125I-rhEGF (*) per gram of jelly, onto full-thickness skin wounds in rats. Values represent the mean  SD of 12 replicates
`per sampling time
`
`Table 1. Cumulative profiles of 125I-rhEGF-associated radioactivity at the lesion sites expressed as percentage of the dose applied
`per gram of tissue (%D/g), after topical administration of both strengths (mean  SD)
`
`Dosage strength (mg/g)
`
`Time (h)
`
`2
`
`4
`
`6
`
`12
`
`24
`
`48
`
`72
`
`10
`20
`
`11.3(  2.8)a
`24.8(  4.3)b
`
`13.8(  5.6)a
`29.7(  5.1)b
`
`20.7(  1.5)a
`36.7(  7.6)b
`
`36.1(  14.5)a
`49.7(  10.8)a
`
`19.1(  5.8)a
`33.1(  8.1)b
`
`5.6(  3.6)a
`26.3(  4.0)b
`
`ND
`14.3(  4.5)
`
`a,bDifferent superscript means significant differences (p5 0.008).
`ND, no drug was detected at 144 h.
`
`tissue
`associated radioactivity per gram of
`(%D/g) at each time point. Statistically signifi-
`cant differences were detected between the two
`strengths at all times evaluated, except for 12 h
`when the differences were not evident despite
`figures suggesting otherwise.
`Interestingly, both jellies showed a rather
`similar kinetic pattern as judged by the shape
`of the curves depicting their respective accumu-
`lation behaviour at the wound site. There was a
`remarkable difference, however, in the extent of
`disposition as seen from their individual AUC.
`Even though the time to peak was the same for
`both formulations (12 h),
`the tissue-associated
`radioactivity peaked to 49.7%D/g for the higher
`strength, while the peak value was only 36.1%
`D/g for the lower one, which showed a sharper
`decline after 24 h, reaching a value of zero at 72 h,
`while its counterpart was still
`radioactively
`detectable.
`
`Certainly, the local application of both jellies
`elicited typical deposition responses for
`the
`experimental animal model used, with less than
`25%D/g being detected during the first 2 h.
`Table 2 shows average kinetic parameters char-
`acterizing each formulation studied. Accordingly,
`the relatively slow elimination rate from the
`wound site is a common feature for both
`strengths, although the 20 mg/g jelly showed a
`more protracted course as indicated by a larger
`MRT, HVD and in situ availability (Fin situ), a fact
`reinforced by the diffusion-limited release pat-
`tern of the jellies. Moreover, we must be aware of
`the capacity-limited (i.e. saturation) non-linear
`kinetic at the lesion site, which was evident when
`comparing the disposition parameters of both
`jellies. An interesting plateau was observed
`between 24 and 48 h for the higher dosage
`strength (see Figure 1), which is probably
`associated with the non-linear pattern.
`
`Copyright # 2004 John Wiley & Sons, Ltd.
`
`Biopharm. Drug Dispos. 25: 193–201 (2004)
`
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`

`

`COMPARATIVE 125I-RHEGF JELLY KINETIC PATTERN
`
`197
`
`Table 2. Mean kinetic parameters characterizing 125I-rhEGF
`topical disposition profiles for each experimental group
`
`Parameter
`
`Jelly 10 mg/g
`
`Jelly 20 mg/g
`
`Time to peak (h)
`Peak level (%D/g)
`AUC0t (%D h/g)
`AUC01 (%D h/g)
`MRT (h)
`HVD (h)
`Fin situ (%)
`RHVD
`Lambda z (h1)
`
`12
`36.1
`825.5
`933.9
`25.4
`21
`62.6
`4.5
`0.052
`
`12
`49.7
`2077.9
`2823.4
`55.7
`50
`94.6
`10.6
`0.019
`
`Parameters were estimated using non- compartmental analysis.
`
`Finally, no measurable levels of radioactivity
`were detected in either the blood or urine
`samples (results not shown) suggesting a negli-
`gible absorption of 125I-rhEGF from the wound
`into the bloodstream.
`
`Discussion
`
`125I-rhEGF Cumulative profiles at the lesion site
`When considering the 125I-rhEGF disposition
`profiles at the lesion site, it is clear that there
`was an initially fast concentration gradient-
`driven release of the drug from the formulations
`up to 2 h after application. Then skin-associated
`counts continued to increase, although less
`abruptly, until reaching the 12 h peak, starting a
`slow and sustained move downward thereafter.
`Further studies are needed to find out whether
`this in vivo release pattern is consistent with an
`earlier and stronger promotion of wound healing
`events.
`In our view, a saturation phenomena could
`slow the in situ clearance rate at the higher dose,
`while the elimination phase suggests non-linear
`kinetic behaviour as corroborated by the esti-
`mated disposition kinetic parameters
`(see
`Table 2).
`Experimental evidence suggests that continu-
`ous receptor occupancy for at least 8–12 h is a
`necessary condition for EGF mitogenic signalling
`[5].
`In a previous study our laboratory reported
`that only about 30% of the administered dose
`
`remained at the lesion site, 4 h after administra-
`tion of either 5 or 10 mg/ml 125I-rhEGF saline
`solutions (delivering 200 or 400 ng of 125I-rhEGF
`per site) in a full-thickness skin lesion model in
`rats. These relatively short mean residence times
`revealed a rapid elimination process as early as
`about 2 h after delivery [7].
`It was hypothesized that a jelly formulation
`could increase 125I-rhEGF permanence at
`the
`lesion site, accomplishing receptor occupancy
`for an appropriate length of time. In this study
`jelly containing two 125I-rhEGF
`400 mg of
`strengths, 10 and 20 mg/g, delivering 4 or 8 mg
`at each lesion site was used. This was signifi-
`cantly higher than those delivered by the saline
`solutions. These jellies are a candidate in the
`selection of a suitable formulation for dermal
`application.
`Figure 1 shows a remarkable persistency of
`drug- associated radioactivity at the lesion site
`over 20 h after application of both strengths. As
`can be seen in Figure 2, the corresponding kinetic
`profiles for both strengths, i.e. 10 and 20 mg/g,
`present a longer course, while Table 2 shows half-
`value durations (HVD) greater than those for
`saline solutions containing lower amounts of the
`peptide (5 and 10 mg/ml). Therefore a clinically
`significant increment in the sojourn time of the
`125I-rhEGF at the lesion site is assumed.
`This topical
`formulation, which is able to
`provide the lesion site with steady amounts of
`125I-rhEGF over the required time period, would
`probably be able to achieve the wound healing
`response needed in terms of cell proliferation,
`collagen deposition and protein synthesis. On the
`contrary, 125I-rhEGF saline solutions with HVD
`ranging from 3.7 to 4.7 h are not expected to meet
`either the qualitative or the quantitative require-
`ments for wound healing, even though the dose
`strength is increased. In accordance with this,
`Jijon et al. [10] found no significant differences
`between pig full-thickness wounds treated either
`with 2 ml of a 300 ng/ml EGF solution (600 ng of
`EGF per wound site) or Ringer lactate solution
`alone, even when the treatment was applied
`twice a day. Significant differences regarding the
`wound tear strength were found, however, when
`the vulnerary agent was administered in the
`form of a gel containing 0.2% carbopol 940
`polymer for the treatment of mice skin incisions
`
`Copyright # 2004 John Wiley & Sons, Ltd.
`
`Biopharm. Drug Dispos. 25: 193–201 (2004)
`
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`

`

`198
`
`J. DUCONGE ET AL.
`
`Figure 2. Comparative in situ disposition patterns of the three earlier studied 125I-rhEGF solutions containing 40 (&), 200 (.) and
`400 ng (n) of 125I-rhEGF and both strengths, 10 (*) and 20 mg of 125I-rhEGF (}) per gram of jelly. Values represent the mean  SD
`per sampling time. Observe the remarkable kinetic difference between either of the solutions and both jellies, particularly with
`regard to the sojourn time at the lesion site
`
`or full thickness wounds compared with saline
`solutions with the same EGF concentration [11,
`12]. It became apparent that the dosage form and
`the formulation composition were key factors in
`the disposition of the drug.
`Accordingly, topical jelly formulations contain-
`ing two 125I-rhEGF strengths, showed HVD (21
`and 50 h, respectively) and RHVD values (4.5 and
`10.6, respectively) suggesting the likelihood of a
`more effective contribution to wound restoration
`processes, considering the requirement for re-
`ceptor occupancy. It is important to note that the
`estimated RHVD values were higher at the lesion
`site than the level for a strong retardation (i.e.
`RHVD53). In this regard, the results are in good
`agreement with the above mentioned criteria for
`the optimal development of a topical rhEGF
`delivery system.
`
`125I-rhEGF Detection in blood and urine samples
`
`Interestingly, measurable levels of radioactivity
`were not detected in either the blood or urine
`samples (results not shown).
`In fact,
`these
`indistinguishable from background counts could
`be associated with an extremely rapid disap-
`pearance from the circulation, which have been
`125I-rhEGF, mainly because of
`described for
`cellular uptake by the liver as well as by the
`red blood cells [13]. On the other hand, so-called
`
`receptor-mediated endocytosis (RME) is recog-
`nized as one of the most important clearance
`mechanisms for biologicals [14]. In this regard,
`the clearance site is at the same time the target
`site. Taking this into consideration, it could be
`the wound-associated in situ
`assumed that
`clearance, following the RME pathway, probably
`accounts for the negligible levels of TCA-pre-
`cipitable radioactivity in either the blood or urine
`samples. Moreover, phagocytic phenomena at the
`wound site should not be discounted.
`
`Local 125I-rhEGF disposition analysis
`Kinetic analysis of 125I-rhEGF disposition profiles
`(see Figure 1) at
`the lesion site, allows the
`assessment of the possible role of either in situ
`125I-rhEGF-associated radioactivity uptake, or
`dermal clearance,
`including receptor-mediated
`endocytosis (RME) with protease-driven clea-
`vage,
`in the dose-dependent
`(i.e. non-linear)
`deposition temporal pattern observed.
`Notably, the classical linearity criteria based on
`direct proportionality relationships between
`AUC and doses were absent. As can be seen in
`Table 2, the estimated kinetic parameters were
`greater than expected for the higher strength.
`Indeed, the AUC was 3-fold greater for a strength
`that was twice as concentrated. Likewise, the
`
`Copyright # 2004 John Wiley & Sons, Ltd.
`
`Biopharm. Drug Dispos. 25: 193–201 (2004)
`
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`

`COMPARATIVE 125I-RHEGF JELLY KINETIC PATTERN
`
`199
`
`MRT was 2-fold larger and the peak level was
`almost 1.5 times higher.
`In comparison, the 125I-rhEGF jelly formulation
`with the higher strength showed a peak level
`(49.7%D/g) that exceeded those previously re-
`ported for 125I-rhEGF saline solutions, at lower
`doses (i.e. 5 and 10 mg/ml of 125I-rhEGF). So, a
`possible explanation for the observed disposition
`behaviour (see Figure 1) could be the saturation
`of the kinetic mechanism involved at the lesion
`site, in a similar manner to that suggested by the
`disposition analysis of 125I-rhEGF saline solu-
`tions. Also, nonspecific binding of 125I-rhEGF-
`associated radioactivity due to the increased
`receptor-unbound 125I-rhEGF fraction probably
`occurs at the lesion site.
`It could be assumed that steady state receptor
`binding conditions were achieved from 6 h
`onwards. Interestingly, the estimated in situ 125I-
`rhEGF availability factors (Fin situ), at the lesion
`site, for the two jellies (10 and 20 mg/g) were
`62.6% and 94.6%, respectively. Nonetheless, the
`AUC (933.9%D h/g) of
`the lower
`strength
`ranged from 6 to 32-fold those for saline solutions
`with doses even 100-fold lower. Similarly, the
`AUC (2823.4%D h/g) for the higher strength
`was 98-fold greater than that of the 125I-rhEGF
`saline solution containing the lower dose. These
`results are probably associated with a slower rate
`of 125I-rhEGF release from the jelly into the
`wound site during the plateau, and also to the
`slower in situ diffusion rate. Note that the HVD
`values always suggested a longer sojourn time at
`the lesion site. Although an overestimation of
`Fin situ values due to the calculation procedure
`used, as a consequence of non-linearity must be
`taken into account.
`Figure 1 shows the typical disposition re-
`sponses at the lesion site for the formulations
`under study, characterized by two superimposed
`the 125I-
`kinetically relevant processes: First,
`rhEGF release by diffusion from the jelly for-
`mulations is the major process during the
`the 125I-rhEGF-associated
`upward portion of
`radioactivity versus time curve. Afterwards,
`clearance processes become the major events
`characterizing the downward phase of the dis-
`position curve. Probably, diffusion of 125I-rhEGF
`into the wound site and its overall elimination
`rate (including RME/ down regulation clear-
`
`the observed disposition
`for
`ance) account
`profiles. However, the incidence of some kinetic
`processes other than the above mentioned cannot
`be rejected, even if they were less important.
`Time to peak (12 h) lagged 10 h behind that of
`the 125I-rhEGF saline solutions (2 h), which could
`be evidence of the slower release rates from the
`jellies compared with the solutions. In our view,
`there are at least two possible hypotheses to
`explain this time lag. First, the slower 125I-rhEGF
`release rates from the jellies, which is a rate-
`limiting step. At
`the same time, a second
`hypothesis emerges from considering the real
`incidence of the dose-dependent non-linearity.
`As a matter of fact, the potential saturation of
`125I-rhEGF binding, at higher doses, should be
`taken into account since the concentration-de-
`pendent nature of the cell-surface EGF receptor
`expression logically suggests it
`is a limiting
`factor. Kinetically speaking, when the rate of
`both release and elimination processes are equal
`then a peak level is achieved, so if the elimination
`rate is reduced due to saturation, a maximum
`should be observed later.
`Alternatively, it is true that formulation addi-
`tives can markedly increase 125I-rhEGF stability
`compared with 125I-rhEGF solutions, in addition
`to yielding higher AUCs,
`suggesting that
`125I-rhEGF may remain in the lesion area for
`longer. Certainly, the most significant 125I-rhEGF
`clearance phenomena at
`the lesion site are
`associated with either down-regulation of the
`cell-surface EGF receptor (i.e. RME) or catabolic
`mechanisms such as protease-driven degrada-
`tion. RME, as part of the physiological regulatory
`events upon EGF receptor trafficking and the
`corresponding
`rhEGF-induced
`intracellular
`downstream signalling pathways, is able to alter
`the 125I-rhEGF-disposition behaviour [14]. Such a
`down-regulation phenomenon has already been
`in situ episode
`recognized as an important
`directly related to the capacity-limited processes
`at the lesion site [7].
`including growth
`Most biological products,
`factors and cytokines, exhibit non-linearity [15,
`16, 17]. Saturation of binding to the cell surface
`receptor could be the major factor causing the
`typical nonlinear elimination profile, which is
`affected by down-regulation events. Accordingly,
`it is still believed that accumulation is possible
`
`Copyright # 2004 John Wiley & Sons, Ltd.
`
`Biopharm. Drug Dispos. 25: 193–201 (2004)
`
`Apotex, Inc. (IPR2019-00400), Ex. 1018, p. 008
`
`

`

`200
`
`J. DUCONGE ET AL.
`
`but it depends on several factors such as EGF
`receptor density and the rate of its regulatory
`events.
`the
`Interestingly, saturation can also alter
`RME-driven clearance mechanism. An important
`factor governing the kinetics of biological drugs
`is the key role played by cell-surface receptors in
`their elimination from the body. In this sense, it is
`logical to expect any change in drug clearance to
`depend on the contribution of the RME process.
`It should be borne in mind that the fraction of
`dimerized receptors, as a function of
`ligand
`concentration, is biphasic or bell-shaped. Accord-
`ingly, any biological event that requires receptor
`dimerization to be activated, such as RME, is also
`predicted to show a dose-dependent bell-shaped
`pattern with self-antagonism at (supra) physio-
`logical concentrations.
`In this regard,
`it was
`assumed that at the higher strength (20 mg/g) a
`considerable amount of monomeric ligand–
`receptor complexes could be present, which are
`unable to enter any clearance pathway using the
`internalization step and hence elimination is
`reduced with subsequent drug accumulation
`and enhanced transit time at the lesion site.
`Because topical products belong to a class
`termed as locally acting drug products, and the
`drug is usually delivered directly to or near the
`intended site of action, measuring the 125I-rhEGF
`uptake into and elimination from the wound site
`can provide a kinetic means of assessing the
`safety and efficacy of the 125I-rhEGF jelly. In this
`setting, a protocol was followed including ki-
`netic/dynamic events at the target site, since
`damaged skin shows high variability and
`changes over time.
`On the other hand, safety and efficacy should
`be documented for all strengths of topical drug
`products under submission. As conventionally
`stated by regulatory issues, some evidence of
`local drug availability should be provided for a
`higher strength, using conventional methodolo-
`gies, and in vivo studies might be waived for
`lower strengths, suggesting that an appropriately
`performed in vitro release study may be suffi-
`cient. The current scientific consensus is that
`in vitro release rates from topical formulations
`may predict local drug availability, especially in
`some instances where the stratum corneum is
`damaged or disrupted [18]. In this context, the
`
`drug release rate may reflect drug delivery direct
`to the dermal target site without passage through
`the stratum corneum and other skin barriers.
`However, considering a full-thickness skin lesion
`model in rats, an in vitro release rate study could
`miss some important in vivo information about
`saturation or capacity-limited events (i.e. non-
`linearity) at the lesion site, particularly if an
`in vitro–in vivo correlation is not appropriately
`documented. In such a case, lower strengths also
`need categorical in vivo evaluation due to the
`concentration-dependent nature of
`these pro-
`cesses ocurring at the lesion site, which are not
`very well predicted through in vitro testing.
`jelly
`It is concluded that these two topical
`formulations provided more sustained amounts
`of 125I-rhEGF over the period of sampling, even
`though further studies need to be undertaken in
`order to elucidate whether such an in vivo
`disposition pattern is consistent with an earlier
`and stronger promotion of wound healing
`events.
`
`Acknowledgements
`
`This work was supported by the Center of
`Genetic Engineering and Biotechnology, Havana,
`Cuba, and in part by Grant 2000/A018 from
`Alma Mater project awards by the University of
`Havana.
`
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