Single-Pass Intestinal Perfusion (SPIP) and prediction of fraction absorbed and permeability in humans: A study with antiretroviral drugs

European Journal of Pharmaceutics and Biopharmaceutics 104 (2016) 131–139

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Research paper

Single-Pass Intestinal Perfusion (SPIP) and prediction of fraction absorbed and permeability in humans: A study with antiretroviral drugs Thaisa Marinho Dezani, André Bersani Dezani, João Batista da Silva Junior, Cristina Helena dos Reis Serra ⇑ Department of Pharmacy, Faculty of Pharmaceutical Sciences of the University of São Paulo, Avenida Professor Lineu Prestes, 580, Bl. 13, 05508-900 São Paulo, SP, Brazil

a r t i c l e

i n f o

Article history: Received 1 October 2015 Revised 17 March 2016 Accepted in revised form 24 April 2016 Available online 27 April 2016 Keywords: Intestinal absorption Permeability Drug transport Paracellular transport Passive diffusion/transport Fraction absorbed

a b s t r a c t In recent years, the prediction of oral drug absorption in humans has been a challenge for researchers and many techniques for permeability studies have been developed for several purposes, including biowaiver processes. The Single-Pass Intestinal Perfusion (SPIP) method performed in rats can provide permeability results closest to in vivo condition. The purpose of the present study was to evaluate the intestinal permeability of the antiretroviral drugs lamivudine, stavudine and zidovudine using the SPIP method in rats and to predict their permeability (Peff,humans) and fraction absorbed (Fa) in humans. Metoprolol and fluorescein were used as marker compounds of high and low permeability, respectively. The effective permeability (Peff) results showed that stavudine and zidovudine have high permeability characteristics while lamivudine presented the lowest result. From Peff values obtained in rats, the Peff,humans and Fa were calculated. The use of SPIP in rats and calculations for absorption prediction in humans may indicate the transport mechanisms and/or pre-systemic metabolism involved on permeation processes of drugs, since this model is the closest to in vivo conditions. Ó 2016 Elsevier B.V. All rights reserved.

1. Introduction Oral administration is the main route for drugs due to convenience, safety, low cost and great acceptance by patients [1]. The efficacy and safety of drugs are associated with bioavailability, which is a biological property related to rate and extent of absorption. Oral absorption depends on drug release from a dosage form (dissolution/solubility) in luminal fluids and its permeability through intestinal membranes. Thus, solubility and permeability processes are fundamental for absorption [2,3]. Based on solubility and permeability characteristics, the Biopharmaceutical Classification System (BCS) was proposed by Amidon and colleagues (1995) and it has been used as a tool for new drugs and new pharmaceutical developments. The BCS classifies drugs into four classes: class I (high solubility and high permeability), class II (low solubility and high permeability), class III (high solubility and low permeability) and class IV (low solubility and low permeability) [2,4,5]. Also, the Biopharmaceutics Drug Disposition Classification System (BDDCS) was proposed as a tool to predict the disposition of a drug. In this system, compounds can be classified into four classes according to their characteristics of solubility and metabolism. The interaction between drugs and transporters can be evaluated as well [6]. ⇑ Corresponding author. E-mail address: [email protected] (C.H.R. Serra). http://dx.doi.org/10.1016/j.ejpb.2016.04.020 0939-6411/Ó 2016 Elsevier B.V. All rights reserved.

In 2000, the Food and Drug Administration Agency (FDA) published a guideline based on BCS for waiver of bioavailability and in vivo bioequivalence studies for class I drugs. For biopharmaceutical classification purposes, the FDA recommends methods that can predict the absorption extension of an oral drug product [3]. Permeability can be directly determined by measuring the rate of mass transfer across human intestinal membrane or indirectly by estimating the extent of drug absorption in pharmacokinetics studies in humans in comparison with reference dose administered by intravenous route. Alternatively, different systems can be used for prediction of oral drug absorption in humans and to classify drugs according to permeability characteristics, such as: artificial membranes and cellular cultures (in vitro methods), ex vivo studies using isolated intestinal segments (ex vivo methods) and intestinal perfusion studies (in situ methods) [3]. The FDA guidance recommends in situ perfusion studies as a tool for classifying a drug according to its permeability characteristics based on the BCS and this method can be applied for biowaiver purposes of drugs orally administered. The method is widely used because it is simple and presents low cost and surgical easiness in comparison with in vivo studies [3,7–10]. Different techniques have been studied for absorption of drugs using in situ perfusion, such as: in situ Thiry-Vella fistula, in situ intestinal single-pass perfusion, in situ intestinal recirculating perfusion, in situ intestinal perfusion with venous sampling, in situ vascularly perfused intestine-liver and in situ Loc-I-Gut [11].

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The Single-Pass Intestinal Perfusion (SPIP) is based on the principle that drug concentration in perfusion solution decreases over time and it is attributed to drug absorption. It allows to determine the rate and extent of absorption through the intestinal membrane [1,7,12,13]. Many studies discuss about the usage of SPIP for absorption prediction in humans. A good correlation is reported in the literature and it is related to the protocol adopted for permeation studies that provides a functional intestinal barrier capable to mimic the physiological conditions, including intact blood supply, innervation and presence of transporters and enzymes. Several compounds with a variety of permeation pathways were tested and a good correlation was observed in different studies involving the SPIP method, with r2 greater than 0.7 [11,14–17]. Also, a good correlation between in situ perfusion and transfected cell line (Caco-2/hPEPT1) was reported in the literature in order to investigate the interaction between a drug actively transported and its carrier (PEPT1) [18]. However, for many drugs, the correlation between cell-based systems and those models that mimic the in vivo situation (as SPIP) may be very difficult to be observed due to irregular expression of transporters and enzymes in in vitro models. In most cases, a good correlation between in vitro and in vivo models is attributed to permeability of drugs that are mainly transported by passive diffusion. In a study conducted by Amidon, Sinko and Fleisher, the correlation between the permeability in rats for estimating human oral fraction dose absorbed was evaluated. In this case, there was a good correlation between the permeability determined by in situ perfusion and the human oral availability, even when compounds transported by passive or carrier-mediated mechanisms were compared [19]. Cao and coworkers (2006) found a good correlation (r2 = 0.8) of effective permeability (Peff) values between rats and humans. A good result obtained from rats to predict absorption is related to the correlation between permeability characteristics in intestinal tissue from rats and intestinal tissue from humans in terms of physiological and morphological similarities [20]. Antiretroviral drugs are widely used for HIV (human immunodeficiency virus) treatment and permeability data obtained from SPIP technique are not reported in the literature, as well as permeability in humans. Moreover, antiretroviral drugs are adopted for some countries for universal access to HIV treatment and understanding about their behavior in the body may direct strategies for dosage form development. Based on these conditions, the purpose of the present study was to evaluate the intestinal permeability of the drugs lamivudine (3TC), stavudine (d4T) and zidovudine (AZT) using SPIP in rats and to predict the permeability (Peff,humans) and fraction absorbed (Fa) in humans.

2. Materials and methods 2.1. Chemicals and reagents Active pharmaceutical ingredient (API) working standards of 3TC, d4T and AZT were obtained from Cristália Produtos Químicos Farmacêuticos (São Paulo, Brazil) and Fundação para o Remédio Popular (FURP, São Paulo, Brazil) and used as received. Fluorescein (FLU) and metoprolol (METO) were used as markers in SPIP studies and were purchased from Sigma–Aldrich (St. Louis, MO, USA). Chromatographic grade acetonitrile, methanol, monobasic potassium phosphate and orthophosphoric acid were purchased from Merck (Darmstadt, Germany). High purity deionized water was obtained from Milli-Q purification system (Millipore, MA, USA). Sodium chloride (NaCl), potassium chloride (KCl), sodium phosphate dibasic (Na2HPO4), sodium phosphate monobasic (NaH2PO4),

mannitol, polyethylene glycol 4000 (PEG-4000) and were used for perfusion solution preparation.

D-glucose

2.2. Perfusion solution Perfusion solution for SPIP assays was prepared with NaCl 48 mM, KCl 5.4 mM, Na2HPO4 28 mM, NaH2PO4 43 mM, mannitol 35 mM, PEG-4000 1 g/L and anhydrous D-glucose 10 mM [21]. All components were added in purified water (Milli-Q) until complete dissolution. As needed, pH was adjusted to 6.5 with HCl or KOH solution [22,23]. The concentration of each drug or marker (Table 1) used in the perfusion studies was determined by dividing the highest prescribed dose by 250 mL. This volume should be taken in order to represent the maximum drug concentration in the intestine [3,7,24]. 2.3. In situ perfusion assays and prediction of permeability and fraction absorbed in humans The use of animals in this study was approved by Ethics Committee under protocol number 235 (Ethics Committee on Animal Use of the Faculty of Pharmaceutical Sciences, University of São Paulo, Brazil). Male Wistar rats weighing 250–300 g were used for all in situ permeability studies (n = 7 for each compound). Animals were fasted for 12 h with free access to water prior to the experiments [7,10,12,21,23–25]. Rats were anesthetized with an intramuscular injection of ketamine–xylazine mixture (0.1 g/kg and 0.02 g/kg, respectively) and placed on heated surface maintained at 37 ± 1 °C. A laparotomy was made through a midline incision of 3–4 cm to expose the small intestine and approximately 10 cm of the proximal jejunum portion was carefully cannulated at both ends. The perfusion solution free of drug at 37 °C (blank perfusion solution) was pumped by peristaltic pump (Minipuls-3, Gilson, France) through the intestine at a flow rate of 0.5 mL/min for approximately 30 min in order to clean out any residual debris. After that, the perfusion solution containing a known concentration of substance (3TC, d4T, AZT, METO or FLU) was perfused at 37 °C through the intestinal lumen at a constant flow rate of 0.2 mL/min. The first 60 min of perfusion period corresponds to steady state, i.e. when the substance is in equilibrium with intestinal membrane [7,24]. After steady state, samples were collected from distal portion of jejunum at 15 min intervals (15, 30, 45, 60, 75, 90, 105, and 120 min) in pre-weighted vials. Samples were immediately frozen at
20 °C until analysis by high performance liquid chromatography (HPLC). At the end of the sampling, animals were euthanized with saturated potassium chloride solution by intracardiac injection, according to protocols for euthanasia in experimental animals [12,26]. After death, the intestinal segment was removed for measurements of length and radius (l and r, respectively).

Table 1 Concentrations of drugs/markers in perfusion solution used for SPIP studies. Drug/marker

Highest prescribed dose (mg)a

Concentration (mg/mL)

3TC d4T AZT METO FLU

300 40 300 100 –a

1.20 0.16 1.20 0.40 0.10

a As FLU is not orally administered, the concentration in perfusion solution was standardized for 0.1 mg/mL in SPIP studies.

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The net water flux (NWF) in the in situ perfusion studies (water absorption and efflux in the intestinal segment) was determined by gravimetric method and the drug concentration in perfusate was corrected for each substance and calculated by the following equation (Eq. (1)) [12,21]:

C out corrected ¼ C out 

Q out Q in

ð1Þ

where Cout corrected = corrected outlet drug concentration, Cout = outlet drug concentration, Qin = inlet perfusate flux (mL/min), Qout = outlet perfusate flux (mL/min). The effective permeability (Peff) was calculated through the equation below (Eq. (2)) [4,15,17]:

Peff ¼


Q in C out corrected  ln A C in

ð2Þ

where Qin = inlet perfusate flux (mL/min), A = intestinal area available for absorption considering radius and the segment length (2 p r l), Cout corrected = corrected outlet drug concentration, and Cin = inlet drug concentration. From Peff obtained in rats (SPIP method), the effective permeability for humans (Peff,humans) was calculated using the equation proposed by Fagerholm, Johansson and Lennernäs (Eq. (3)) [14].

Peff;humans ¼ 3:6 P eff rats þ 0:03  10
4

ð3Þ

where Peff,humans = effective permeability predicted for humans, Peff rats = effective permeability obtained in rats using SPIP method. The fraction absorbed (Fa) in humans can be calculated for each substance using the equation presented by Sugano and colleagues (Eq. (4)) [17,19,27–29]:

F a ¼ 1
expð
38450  P eff rats Þ where Fa = fraction absorbed predicted for humans, Peff effective permeability obtained in rats by SPIP method.

ð4Þ rats

=

2.4. Chromatographic conditions and sample analysis

Samples from in situ perfusion assays were analyzed by a HPLC method previously validated. Parameters as linearity, precision, accuracy, limit of detection, limit of quantification and stability were evaluated [31,32]. The concentration range for calibration curves were: 10.0– 200.0 lg/mL (3TC), 0.5–50.0 lg/mL (d4T), 10.0–200.0 lg/mL (AZT), 25.0–1000.0 lg/mL (METO) and 0.25–25.0 lg/mL (FLU), respectively. Linearity, precision and accuracy were satisfactory (R > 0.999, coefficient of variation and relative error <5%). All validation parameters evaluated were in accordance to ICH guidance and the method was adequate for samples quantification. 2.5. Statistical analysis Statistical comparison of permeation parameters were performed using analysis of variance (ANOVA) and Tukey tests (p < 0.05 was considered as statistically significant) [21,25]. 3. Results and discussion Preliminary studies showed that there was no considerable adsorption of drugs on the components of the pump used for permeability studies. For this evaluation, a comparison between the drug concentration before pumping system and after pumping system was made in order to verify the loss of drug by adsorption. The quantification was made using HPLC system and no unspecific binding was noticed. Also, the stability of the substances was assessed by incubation at 37 °C for 12 h in biological samples and no significant degradation was observed during this period, as shown in Table 3. In both cases, the variation observed was lower than 5%. Care in cannulation procedures of intestinal segment and control of perfusion flow (0.2 mL/min) were taken to preserve blood supply and innervation and to ensure adequate physiological conditions. 3.1. Peff of marker substances

A HPLC system (Merck-Hitachi LaChromÒ, USA) consisting pump (L-7100), autosampler (L-7200), column oven (L-7300), fluorescence (FL) detector (L-7485), ultraviolet (UV) detector (L-7400) and vacuum degasser (L-7612) was used for all quantitative analysis. The system was connected through a D-7000 interface to specific software in a computer for data collection and processing (Chromatographic station software, HPLC system manager, version 4.1, 1994–2001, P/N 810866201). Table 2 summarizes all chromatographic conditions used in this study for quantification of 3TC, d4T, AZT, METO and FLU in permeability studies. The chromatographic conditions in this study were adapted from methods described in the literature [24,30,31].

FLU, a low permeability marker, was used to evaluate integrity of the intestinal membrane. This marker is widely used in different Table 3 Accuracy and precision evaluated for stability of antiretroviral drugs in perfusion solution at pH 6.5/37 °C for 12 h. Drug

d4T 3TC AZT

0h

12 h

Accuracy (%)

Precision (%)

Accuracy (%)

Precision (%)

103.2 104.1 99.8

99.8 100.2 101.1

101.2 103.6 98.2

98.4 100.1 96.2

Table 2 Chromatographic conditions used for quantification of 3TC, d4T, AZT, METO and FLU samples in SPIP assays. Chromatographic conditions

3TC, d4T and AZT

METO

FLU

Detector Wavelength (nm)

UV 270

UV 254

Mobile phase

Phosphate buffer 20 mM, acetonitrile and methanol (90:7:3) pH 4.5 PhenomenexÒ Gemini C18 150 mm  4.5 mm 35 30 0.7

Phosphate buffer 20 mM, methanol, acetonitrile, triethylamine (69:15:15:1) pH 4.5 PhenomenexÒ Gemini C18 250 mm  4.5 mm 30 30 1.0

FL Exa: 485 Emb: 515 Phosphate buffer 20 mM, methanol and acetonitrile (40:30:30) pH 4.5 PhenomenexÒ Gemini C 18 250 mm  4.5 mm 35 30 1.0

Stationary phase Oven temperature (°C) Injection volume (lL) Flow (mL/min) a b

Excitation wavelength. Emission wavelength.

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models of permeability studies including Caco-2, artificial lipid membranes and rat jejunum [24,30,33–35]. In this study, FLU presented the lowest permeability value and it is in accordance to works published in the literature (Table 4). As FLU presents negligible or low permeability, low values were expected, otherwise it may be an indication of loss of membrane viability/integrity, compromising the reliability of data [36]. Furthermore, the permeability result of FLU in this study indicates that in situ perfusion method can be used for permeability assays of drugs due to good viability and the guarantee of presence of drug into intestinal segment, i.e., there was not drug leakage out from intestine. As presented in Table 4, permeability values of FLU found in the literature were greater than permeability value obtained in this study. Thus, the in situ perfusion method is suitable for permeability studies due to maintenance of in vivo conditions, innervation and blood supply as well as adequate permeability through paracellular route, presence of mucus layer, expression of active carriers and enzymes [28,39]. For other methods, as ex vivo or in vitro assays, membrane conditions and viability can be evaluated by FLU transport assay. However, the high values of permeability may be related to loss of membrane integrity, especially when these studies are performed for more than 2 h of duration [40]. FLU has paracellular via as transport mechanism, although some studies in the literature have shown other mechanisms involved in the permeation process of this marker. Paracellular route is the reason for the low permeability of some compounds. This via represents less than 1% of surface area available for absorption in the gut [33,35,41–43]. The highest result of FLU in the Table 4 can be explained based on the conditions adopted for the assay [30]. In the study conducted by Dezani and coworkers, the ex vivo model was used for permeability assessment of drugs using FLU as low permeability marker. The comparison between FLU and metoprolol (used in that case as high permeability marker) showed that FLU presented a very low permeability in comparison with metoprolol (79.9  10
5 cm/s). For that particular case, within those experiment conditions as oxygenation, device type, membrane, agitation of donor and acceptor media, lead to the results presented by the authors. Based on this observation, each method can be standardized to be used as a method for permeability studies, as long as a low permeability marker and high permeability marker can be able to differentiate the characteristics of low and high permeability, respectively. For that study, permeability results below FLU may lead to a conclusion that the drug tested has a negligible permeability [30]. Additionally, METO was used as high permeability marker in SPIP permeation studies. Different results of Peff can be obtained from the literature regarding several permeability techniques, as shown in Table 5. Despite differences between published values, Peff result of METO in this study was very close to that one described by Salphati and colleagues (Peff = 5.90  10
5 cm/s) in in situ perfusion assay performed in rats [15]. Results published by other researchers using different methods are relatively close to the value obtained in this study, as 3.30  10
5 cm/s [10], 3.40  10
5 cm/s [44], 4.10  10
5 cm/s [45] and 3.50  10
5 cm/s [46]. These results confirm the high permeability of METO, which is a marker that has passive transcellular pathway as the main transport mechanism. The permeation of METO through the intestinal membrane occurs steadily, without saturation of transporters present in the intestinal membrane [42,43]. As shown in Table 5, Peff values of METO in humans are also described in the literature. The difference between this study and values reported is the intestinal exposure area. Besides,

physiological parameters and test conditions can cause some changes in the results. However, a preliminary evaluation is complicated due to difficulty in determining a boundary value for high and low permeability. Furthermore, each method has particular characteristics that should be considered and a suitable comparison depends on results of markers to define high and low permeability. Thus, conclusions about permeability of drugs (when results are obtained from different techniques and there are no data regarding to marker substances) ends up leading to incomplete or misleading discussions/conclusions. An appropriate approach is to consider the statistical evaluation between results from the same method to assess whether the technique is able to differentiate a low permeability marker from a high permeability marker. Based on these results, FLU and METO indicate that SPIP method used in this study is suitable for permeability assessment of drugs under conditions adopted and it can differentiate the permeability characteristics of the marker substances, according to statistical analysis carried out (Fig. 1). Therefore, the intestinal membrane used in the in situ perfusion method presented intact blood supply and innervation, adequate physiological parameters and enzymes/ proteins active during all experimental period. Based on data presented in Tables 4 and 5, results in Caco-2 are different compared to results obtained in in situ perfusion studies described in this manuscript. For FLU, the lowest value was found in in situ perfusion (0.005  10
5 cm/s), which is expected since this drug has a negligible permeability and the viability of intestinal tissue is kept intact during all time of the experiment. For cellbased studies or ex vivo models, the viability is crucial to keep the adequate conditions in order to preserve the accuracy of results, which is difficult to get since several parameters should be taken into consideration, such as: adequate oxygenation, tissue manipulation, co-solvent use and adequate paracellular route. The comparison between models that mimic in vivo condition (such as in situ perfusion) and those in vitro methods (as cell-based systems) is difficult in some cases. If a high permeability drug is evaluated in in situ perfusion method, its result of permeability tends

Fig. 1. Peff results of 3TC, d4T, AZT, METO and FLU obtained from in situ perfusion study. The error bars represent the SD for each substance (⁄p < 0.000 compared to FLU; ⁄⁄p < 0.001 compared to METO).

Table 4 Permeability and standard deviation (SD) of FLU (n = 7) obtained in this study through in situ perfusion method and values reported in the literature. Permeability ( 10
5 cm/s) ± SD In situ perfusion (this study)

Caco-2 cells (in vitro)

Isolated intestinal membrane (ex vivo)

0.005 ± 0.001

0.02 [34] 0.53 [33] 0.04 [35]

0.86 [33] 0.95 [37] 1.57 [38] 5.4 [30]

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Table 5 Permeability result of METO (n = 7) and its standard deviation (SD) obtained in this study through in situ perfusion method performed in rats and permeability values reported in the literature. Permeability ( 10
5 cm/s) In situ (this study)

In situ (in rats)

In situ (in humans)

Caco-2 (in vitro)

MDCK (in vitro)

Intestinal membrane (ex vivo)

5.32 ± 0.54

1.70 [47] 3.30 [10] 1.47 [7] 15.21 [49] 5.90 [15]

13.40 [4] 15.21 [46]

3.40 [44]

4.10 [45]

3.50 [46] 2.40 [48] 79.9 [30]

to be similar to that one obtained in in vitro method (as Caco-2, for example). In this case, the interaction between the drug and the membrane occur readily and the interference of villi/microvilli is not noticed on the results. But for low permeability drugs, the interaction between the compound and the membrane takes a long time and the permeability results in in situ perfusion tend to be greater in comparison with in vitro methods for those compounds there are not transported by paracellular route. These observations are based on the idea that the anatomical surface area is greater than the absorption surface area in in vivo models, while the anatomical surface area is similar to the absorptive surface area in in vitro models. Thus, the comparison between results from in vivo and in vitro tend to be similar for high permeable drugs, but for low permeable drugs, the permeability results tend to be different due to differences on area available for absorption [50]. Also, the tight junctions in Caco-2 tend to be tighter than rat intestine and since FLU has paracellular route of transport, low values of permeability might be expected for cell cultures. However, the high values of FLU described in the literature (Table 4) can be explained based on the tissue viability and adequate oxygenation. 3.2. Peff of antiretroviral drugs After results analysis of FLU and METO, the intestinal permeability using SPIP method was performed for drugs 3TC, d4T and AZT. Fig. 1 represents graphically the comparison between Peff results of each drug and marker substances and Table 6 shows the Peff results and standard deviation (SD) for all compounds (3TC, d4T and AZT) and data reported in the literature. In a comparative analysis between markers and drugs, d4T and AZT have values of Peff close to METO and far from FLU. 3TC presented the lowest value of permeability in comparison with d4T and AZT but it remains away from FLU result (Fig. 1). The statistical analysis showed significant differences between Peff of FLU and METO. This analysis also indicated that results of 3TC, d4T and AZT are significantly different from substances used as markers, but closest to METO. As shown in Table 6, results obtained from this study are greater than values obtained by other methods. Data from the

Table 6 Peff results and SD obtained from SPIP experiments for antiretroviral drugs (n = 7) and data reported in the literature. Peff  10
5 (cm/s) ± SD in this study

In situ perfusion

Model

d4T

3TC

AZT

Rat intestine

3.96 ± 0.47

3.08 ± 0.26

4.17 ± 0.43

Permeability  10
5 (cm/s) data from literature Method

Caco-2

0.45 [51]

0.10 [52]

MDCK Ex vivo PAMPA

– – –

0.01 [52] 45.8 [30] 0.47 [54]

6.14 2.16 0.17 53.4 –

[52] [53] [52] [30]

literature are not easy to find for those antiretroviral drugs, even in other models. Some results published by researchers related to permeability of antiretroviral drugs were obtained through other methods, but not through in situ perfusion study. Based on this observation, differences on permeability related to transport study may not be noticed. Under conditions adopted in this study, the results are different from those reported in the literature and it can be attributed to specific characteristics of the techniques used. The AZT result in this study is relatively close to the value published by Souza and colleagues using Caco-2 cells (6.14  10
5 cm/ s), which may suggest that the transport occurs by transcellular passive diffusion and the compound may interact with transporters [52]. On the other hand, results in MDCK show a lower permeability, but in that study, MDCK is a canine origin cell line and the expression of transporters is underestimated [52]. Yu and Sinko published a value of 2.16  10
5 cm/s using Caco-2 cells [53]. Dezani and colleagues published a value of 53.4  10
5 cm/s and this values is greater than the expected [30]. However, in this case, the conditions adopted were peculiar as the high speed of magnetic agitation. It is worth noting that methods involved are different and results from cell-based models can present high variability due to some factors such as: source of tissue (adenocarcinoma – Caco-2 or canine kidney – MDCK), expression of influx or efflux carriers, lack of physiological parameters, lack of mucus layer, passage and viability of cells, composition and pH of buffer [1,55]. Then, the comparison between the AZT and data published in the literature can indicate that this drug may be transported by passive mechanism through intestinal membrane, at least as main transport route. However, Pretorius and Bouic and Quevedo and Briñon consider that AZT can be transported by some kind of active mechanism [56–58]. On the other hand, a comparison between two formulations of AZT showed no statistical differences in Cmax, AUC0–t and AUC0–1 (100 mg capsules) for a bioequivalence and pharmacokinetic study carried out in 24 volunteers, which enhance the idea that AZT is passively transported. However, the standard deviation on those results can indicate the involvement of carriers in permeation processes possibly due to interindividual differences [59]. In a similar way, analysis of 3TC results indicates that this drug presented the lowest value in comparison with other substances studied. Reis and colleagues published a study where the permeation mechanism of 3TC is compared between PAMPA, ex vivo and in situ methods. Although the Peff result has not been shown (3.08 ± 0.26  10
5 cm/s), these values were calculated considering Kvf = 5 (for rats) to allow the comparison with other methods [24]. As described in the literature, 3TC and d4T can be transported by carriers, which are active in in situ perfusion model. Furthermore, in SPIP method all physiological conditions are preserved, as mucus layer, innervation, endocrine system, lymphatic and blood supplies and expression of active carriers and enzymes [28,39]. An important factor to be considered is the unstirred water layer (UWL), which corresponds to a layer of water, mucus and glycocalyx more or less static adjacent to the membrane wall and it is

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formed by incomplete mixing of the luminal contents and acts as a major barrier to absorption [55,60,61]. According to some studies, the in vitro and ex vivo models have the UWL thicker in comparison with in vivo condition [4,62] and the UWL can change according to the type and speed of agitation used in the experiment. In in situ perfusion model, UWL (as well as the membrane integrity) is maintained within the limits of the infusion flow in the intestine (0.1– 0.3 mL/min) to avoid disturbance in the luminal compartment, which can lead to changes on permeability results [27]. As previously discussed, the mechanisms of transport for drugs across biological membranes have to be taken into consideration. In this study, METO is transported by passive transcellular, so the results in different methods tend to be similar. FLU is transported by paracellular route and variations on tight junctions and excessive tissue handling may cause discrepancies on results that are not expected in SPIP studies since the conditions are preserved. 3TC, d4T and AZT are transported by passive transcellular route with possible participation of carriers [18,29,42,43,56,57,63–66]. Thereby, different permeability results can indicate differences on transport mechanisms, especially when permeability results from different methods are in disagreement, as shown in Table 6 (for 3TC and d4T results). A screening protocol for novel drugs can direct the use of adequate method for permeability studies. However, the drawbacks presented by each method must be considered and the results may be misinterpreted. Thus, the differences between results obtained in this study and data from literature reveal the difficulty to develop a unique standardized protocol for permeability determination. The SPIP method is useful for permeability assessment of drugs because the transport mechanisms can be better understood and research about metabolic processes may be conducted with great precision in order to clarify the disposition of drug in vivo. For those antiretroviral drugs studied, the good correlation between them is mainly due to passive transcellular transport, although some studies reported in the literature indicate the involvement of carriers. For d4T, studies indicate the involvement of N2 and, possibly, N3 transporters and/or facilitated non-sodium pathways of absorption and involvement of nucleoside transporter should be investigated [67]. For 3TC, hOCTs types 1–3 uptake mechanisms and efflux transport can be involved [24,66]. For AZT, the interaction with P-gp is weak and some studies indicate transport by Na+/nucleoside carrier and a combination of uptake mechanisms [30,56–58]. 3.3. Permeability classification of drugs For this study, the criteria adopted for permeability classification followed the Zakeri-Milani and colleagues paper [68]. The classification mentioned on that work divides drugs into two categories: low permeability (Peff lower than 2  10
5 cm/s) and high permeability (Peff greater than 3  10
5 cm/s), which seems to be adequate for classification of drugs when permeation is evaluated from in situ perfusion studies in rats [68]. However, based on this classification, drugs between 2.0 and 3.0  10
5 cm/s have no designation for their classification, as moderate permeability or boundary permeability. The reason why Zakeri-Milani’s classification was chosen is because the method adopted for permeability studies is very similar to our method (single-pass intestinal perfusion in rats), which is very difficult to find in the literature. Moreover, Zakeri-Milani’s criteria were built to establish a cutoff limit for permeability classification. As the focus of our study is the evaluation of permeability of antiretroviral drugs d4T, 3TC and AZT, the experiments were performed using METO as high permeability marker and FLU as low permeability marker to ensure the method is suitable for permeation studies of these compounds. Also, for the establishment of

a new range for permeability classification, the use of a pool of drugs is necessary. Based on our results, the single-pass intestinal perfusion is suitable to be used for absorption prediction in humans as well as the low permeability drugs, since some parameters are intact in this model, such as: morphological characteristics as villi and microvilli, innervation, blood supply, transporters, enzymes, as mentioned before. None of the other models available for permeability studies can mimic these conditions and in most of the cases, the correlation with in vivo studies is difficult as previously discussed. The permeability study with antiretroviral drugs using the in situ perfusion method as described in our work has the objective to contribute for understanding of permeability through intestinal membrane and use of these data for absorption prediction in humans, which is not reported in the literature. Using the classification presented by Zakeri-Milani and colleagues, d4T and AZT could be considered as high permeability substances in this study. Regarding 3TC, although it presented permeability value close to the limit proposed by these authors, the high permeability characteristic is not discarded for this drug. Therefore, the real classification of 3TC is difficult, considering the standard deviation and other transport mechanisms that may be involved in the absorption processes [24,30,68].

3.4. Prediction of intestinal permeability and fraction absorbed in humans The Peff results obtained by the SPIP method in rats can be used to predict the permeability in humans using the equation proposed by Fagerholm, Johansson and Lennernäs (Eq. (3)) [14]. For METO, some studies have reported Peff obtained from in situ perfusion in humans and the values are: 15.21  10
5 cm/s [46] and 13.40  10
5 cm/s [4] (Table 4). As shown in Table 7, the predicted value of Peff in humans for METO using the Eq. (3) was 19.45  10
5 cm/s, which is a value relatively close to that one reported in the literature. Although similar, differences between humans and rats may exist. It is known that values of Peff are greater in humans due to surface area available for absorption and morphological/physiological conditions, as shown in equation used for drug permeability prediction in humans. Thus, it is normal that values obtained in rats increase when the equation is applied to predict permeability of drugs in humans (Table 7). A study published by Sugano and colleagues proposes an equation that can be used to predict the fraction absorbed (Fa) in humans using Peff rats results and some studies reported in the literature were based on their work [27,28]. Thus, the present study used the equation to predict the fraction absorbed in humans for 3TC, d4T, AZT and METO. Table 8 shows the Fa values predicted for humans from Peff rats (calculated values) and Fa reported in the literature. The comparison between Fa in humans reported in the literature and the Fa predicted may indicate specific characteristics of drugs during permeation process, such as: if a drug is transported

Table 7 Values of Peff predicted in humans based on Peff results performed in rats using SPIP method in this study and the equation proposed by Fagerholm, Johansson and Lennernäs (1996). Drug

Predicted Peff  10
5 (cm/s)

3TC d4T AZT METO FLU

11.39 14.56 15.31 19.45 0.32

T.M. Dezani et al. / European Journal of Pharmaceutics and Biopharmaceutics 104 (2016) 131–139 Table 8 Fa predicted for humans using Peff results obtained from rats in this study and Fa in humans reported in the literature. Compound Fa (%) predicted in humansa

Fa (%) in humans reported in the literature

3TC d4T AZT METO

80 86 98 95

69.4 78.2 79.9 87.2

[69] [70] [71] [27,72–74]

a The Fa predicted of drugs was obtained from the equation proposed by Sugano et al. [27] using Peff results from rats (SPIP method in this study).

137

and the tools for biopharmaceutical classification of drugs have been widely applicable to predict drugs disposition. It can, eventually, aid on the development of new drugs and dosage forms, which contributes for improvement of therapeutic decision. The Peff results of drugs showed that d4T and AZT can be considered as high permeability compounds, while 3TC, due to proximity to the cutoff values proposed by Zakeri-Milani and colleagues, present moderate to high permeability [68]. However, the lack of boundary values to differentiate high/low permeability substances in SPIP studies suggests that more experiments are needed. Prediction of Peff and Fa in humans were calculated and the results showed that SPIP method may be used to predict the absorption of drug indirectly, although some additional tests with more compounds are recommended. Although in vitro methods, such as PAMPA, Caco-2 or MDCK, present high throughput compared to isolated tissues, these models are less predictive and are used for preliminary screening of drugs. Low throughput methods, such as the SPIP method performed in rats, are more predictive and can be used for a secondary screening, mechanistic studies, absorption elucidation, and for assessing the potential efflux carrier interaction of drug candidates because this system presents similarities to in vivo condition [30,75].

Acknowledgements Fig. 2. Fa predicted for humans using Peff results from rats (this study) versus Fa in humans reported in the literature for 3TC, d4T, AZT and METO.

by a specific carrier or if a drug suffers pre-systemic metabolism. In both situations, the Fa of the drug can be lower and, as a consequence, its bioavailability will be incomplete. As presented in Table 8, no data are shown for FLU because this substance is not therapeutically used for oral administration and Fa in humans is not reported in the literature. The Fig. 2 represents the values of Fa predicted in humans calculated from Peff obtained in rats (this study, using Eq. (4)) and Fa described in the literature for each drug. As shown in Table 8 and Fig. 2, the drugs have a good correlation (r2 = 0.7) between Fa calculated and Fa reported. Thus, the Fa equation presented by Sugano and colleagues can be applicable to predict human Fa of drugs, although the inclusion of more drugs would enhance the use of this equation to predict the fraction absorbed of the compounds [27]. This prediction can provide results closest to in vivo data for compounds passively transported. However, a low Fa may be related to involvement of some specific transport mechanism, as influx or efflux carrier. Also, the metabolism that occur in enterocyte must not be ignored because the Fa and, as a consequence, the bioavailability may be incomplete. Calculated Fa values of the all compounds were lower than Fa obtained in humans (data from literature), as shown in Table 8. Although transcellular pathway is the main route for absorption of drugs, paracellular route and carriers can be involved on transport mechanisms of antiretroviral drugs as well as intestinal metabolism [63,65–67]. 4. Conclusion The in situ intestinal perfusion in rats is simpler, cheapest and faster when compared to in vivo studies in humans. Furthermore, rats are widely used to predict permeability in humans because they are suitable to predict oral drug absorption. In addition, a good correlation between rats and humans has been published in the literature due to physiological and morphological similarities between these species. Recently, the biowaiver process has evolved

The authors wish to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil, grant 473329/2009-3 and fellowship), the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Brazil, scholarship grant 2011/09670-3) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil, fellowship) for financial support and the Fundação para o Remédio Popular (FURP, Brazil) and Cristália Produtos Químicos Farmacêuticos for donating the chemicals substances (d4T, 3TC and AZT).

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