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The solubility and dissolution rate of active ingredients are of major importance in preformulation studies of pharmaceutical dosage forms. In the present study passively absorbed drugs are classified based on their intrinsic dissolution rate (IDR) and their intestinal permeabilities. Intrinsic dissolution rate was determined by measuring the dissolution of a non-disintegrating disk of drug and effective intestinal permeability of tested drugs in rat jejunum was determined using single perfusion technique. The obtained intrinsic dissolution rate values were in the range of 0.035-56.8 mg cm-2 min-1 for tested drugs. The minimum and maximum intestinal permeabilities in rat intestine were determined to be 1.6Ã-10-5 and 2Ã-10-4 cm/sec respectively. Four classes of drugs were defined , i.e. Category I: Peff,rat > 5Ã- 10-5 (cm/sec) or Peff,human > 4.7Ã- 10-5 (cm/sec), IDR > 1(mg/min/cm2), Category II: Peff,rat > 5Ã- 10-5 (cm/sec) or Peff,human > 4.7Ã- 10-5 (cm/sec), IDR < 1(mg/min/cm2), Category III: Peff,rat < 5Ã- 10-5 (cm/sec) or Peff,human < 4.7Ã- 10-5 (cm/sec), IDR > 1(mg/min/cm2) and Category IV: Peff,rat < 5Ã- 10-5 (cm/sec) or Peff,human < 4.7Ã- 10-5 (cm/sec), IDR < 1(mg/min/cm2). According the obtained results and proposed classification for drugs, it is concluded that drugs could be categorized correctly based on their intrinsic dissolution rate and intestinal permeability values.
Keywords: Intrinsic dissolution rate, intestinal Permeability, Biopharmaceutics.
The solubility and dissolution rate of active ingredients are of major importance in preformulation studies of pharmaceutical dosage forms (1-6). The formulation characteristics including shelf life, process behavior, and even the bioavailability are affected by physicochemical properties of drug molecules (7). The intrinsic dissolution rate (IDR) has been used to characterize solid drugs for many years (8, 9). For example it could be used to understand the relationship between the dissolution rate and crystalline form and also to study the effects of surfactants and pH on the solubilization of poorly soluble drugs (10, 11). IDR is generally defined as the dissolution rate of a pure drug substance under the condition of constant surface area, agitation or stirring speed, pH and ionic strength of the dissolution medium. The true intrinsic dissolution rate may be better described as the rate of mass transfer from the solid surface to the liquid phase. The apparatus for intrinsic dissolution testing was originally developed by John Wood which enables the calculation of the dissolution rate per centimeter squared of the intrinsic ingredients of pharmaceutical products (12-14). It has been suggested that it might be feasible to use IDR to classify drugs instead of solubility (9). The reason is that, just like permeability, IDR is a rate phenomenon instead of an equilibrium phenomenon. Therefore it might correlate better with in vivo drug dissolution rate than solubility, although for drugs having either extremely high or low dose, discrepancies may exist between the solubility and IDR methods (9) since dose is considered in the classification of solubility while intrinsic dissolution does not consider the effect of dose. On the other hand there are a number of in vitro and in situ experimental models have been developed which determine the intestinal absorptive potential of a drug and the mechanism of absorption (15, 16). Among these methods single-pass intestinal perfusion (SPIP) approach is the most frequently used technique which provides conditions closer to what is faced following oral administration. SPIP technique possesses a preserved microclimate above the intestinal membrane which makes it less sensitive to pH variations. This technique provides the unique advantages of experimental control (compound concentration and intestinal perfusion rate) and the ability to study regional differences; factors that may influence the intestinal absorption of a compound. In the present study the intrinsic dissolution rate and rat intestinal permeability (using SPIP technique) were measured for drugs with different physicochemical properties. The suitability of IDR-permeability for biopharmaceutical classification of drugs was evaluated.
Materials and methods
Naproxen, atenolol, metoprolol, propranolol, verapamil hydrochloride and ibuprofen were provided from Shasun (Shasun Chemical & Drugs LTD, India). Ketoprofen and antipyrine were from Sigma (Sigma, Canada) and Hoechst (Hoechst, Germany) respectively. Furosemide and hydrochlorothiazide was provided from Fls (Fls, Italy). Ranitidine and cimetidine were obtained from Uquifa (Uquifa, Spain) and piroxicam was from Ciba-Geigy (Barcelona, Spain). Monobasic potassium phosphate (KH2PO4) and sodium hydroxide (NaOH) were purchased from Merck (Darmstadt, Germany).
Procedure of IDR measurement
A quantity of 100 mg of each drug was compressed at an average compression force of 7.84 MPa for 1 minute to make non-disintegrating compacts using die and punch with diameter of 6 mm. In order to prevent capping, in the case of piroxicam and carbamazepine the compression force were 1.96 and 19.6 MPa respectively. The surface area of the compacts was 0.2826 cm2. The improved method of wood et al was used for disk dissolution studies (14). Compacts were placed in a molten beeswax-mold in such a way that only one face could be in contact with dissolution medium. Dissolution study was conducted using USP II dissolution apparatus using 900mL of phosphate buffer (pH=6.8) at temperature of 37Â°C Â± 1Â°C as the dissolution media with paddle rotating at 100 rpm. Samples were collected through 0.45-Âµm syringe filters over a period of 8 hours for low-soluble and 20 minutes for highly soluble drugs. Sampling time intervals were 30 min and 2 min respectively. All studies were carried out in triplicate. Absorbances were determined in triplicate using a UV-Vis spectrophotometer (UV160, Shimadzu, Kyoto, Japan) at the maximum absorbance wavelength for each active tested (Table 1). The cumulative amount dissolved per surface unit of the compact was plotted against time for each vessel. The slope of the linear region (R2â‰¥ 0.95) was taken as intrinsic dissolution rate. IDR is easily calculated by
G = (dw/dt)(1/S) = DCs/h Eq.1
where G is intrinsic dissolution rate (mg/min/cm2); dw is the change in drug dissolved (mg); dt is the change in time (minutes); S is the surface area of the compact (cm2); D is diffusion coefficient (cm2/sec); Cs is solubility (mg/cm3) and h is stagnant layer thickness (cm).
Solubilities were determined in at least triplicates by equilibrating excess amount of drugs in phosphate buffer solutions (pH=6.8). The samples were kept in thermostated water bath at 37Â°C and shaked at a rate of 150 rpm for 24 hours. The absorbances of filtered and suitably diluted samples were measured with an UV-VIS spectrophotometer ((UV160, Shimadzu, Kyoto, Japan) at the maximum absorbance wavelength for each active tested. The solubilities were calculated using calibration curves determined for each drug.
Measurement of rat intestinal permeability coefficients:
The anesthesia and surgery were performed in accordance with a previously validated in situ intestinal perfusion method in rats (17). Details of the procedure, analytical methods and also permeability coefficient calculation were explained in authors published works (16, 18, 19). Briefly, male Wistar rats (weight, 250-300 g; age, 7-9 weeks) were maintained on 12 h light- dark cycle and fasted 12-18 h before experiment. On the day of experiment a single pass constant flow (2 ml/min) of drug containing perfusate (PBS pH=7.2, 37oC) was established through the ligated rat jejunal segment and the outlet samples were collected every 10 min in microtubes up to 90 min and stored at -20oC until analysis. Finally the animal was euthanitized with a cardiac injection of saturated solution of KCl. In all animal studies "Guide to the care and use of experimental animals" by Canadian Council on Animal Care, was followed (20). Permeability values were calculated using following equation according to the parallel tube model:
Peff = -Q ln(Cout(corrected)/Cin)/2Ï€rl Eq.2
In which Cin and Cout are the inlet and outlet concentrations of compound respectively. Cout is corrected for volume change in segment using phenol red concentration in inlet and outlet tubing. Q is the flow rate (0.2 ml/min), r is the rat intestinal radius (0.18 cm) and l is the length of the intestinal segment (16, 21).
Current BCS guidance defines an API as "highly soluble" when the highest dose recommended is soluble in 250 mL or less of aqueous media over the pH range of 1.2 to 7.5 (22). However the pH 6.8 is scientifically justified over pH 7.4 (22, 23). In order to set a condition for BCS classification of compounds and since small intestine is the major site for drug absorption, where the pH is about 6.8, IDR measurements were conducted in pH 6.8. Table 1 shows the solubility of model drugs at 37Â°C in pH=6.8. The high solubility of ranitidine in experimental condition and reaching viscose solution in high concentrations made it difficult to assay its exact solubility (24). The determined intrinsic rates of dissolution are also given in Table 1. The presence of sink condition in dissolution medium during the experiment is upholded by comparison of the final concentration of drugs and their solubility in dissolution medium. As seen in the Table 1, the ranking order of aqueous solubility and IDR are almost but not exactly the same. In general, compounds with high solubility exhibited IDR of greater than 3 mg/cm2/min but compounds with low solubility had IDR of less than 1 mg/cm2/min. However antipyrin which showed greatest IDR amongst the tested compounds, was determined to be less soluble than ranitidine and metoprolol. Furosemide exhibited higher IDR but slightly lower solubility in comparison to naproxen. In the same way piroxicam exhibited higher IDR but slightly lower solubility in comparison to carbamazepine. This could be explained by the fact that unlike solubility, IDR is a rate phenomenon and besides solubility is dependent on wetability and diffusivity of the compound (25).
The observed intestinal permeability values in rats for tested drugs (16) are also presented in Table 1. For comparison the respective intestinal permeability and fraction of dose absorbed in human are also cited. Classification of tested drugs based on their intestinal permeability and IDR for human and rat is shown in Fig. 1 and Fig. 2 respectively.
Drugs are scientifically identified based on their solubility and human intestinal permeability (26). The biopharmaceutics classification system (BCS), consists of four drug categories: class I (highly soluble and highly permeable), class II (low soluble and highly permeable), class III (highly soluble and low permeable) and class IV (low soluble and low permeable). Since human intestinal permeability could be predicted with precise using the rat effective permeability values (16), the same classification can be constructed utilizing the solubility and rat intestinal permeability values (27). IDR is a parameter which could be used easily to characterize the pure drug substance. The determination of this parameter allows labs to screen experimental drug formulations and to understand their behavior under different bio-physical conditions. Table 1 shows the obtained solubility and IDR values in the present work for tested drugs. The obtained IDR values on the tested drugs in the present work are slightly greater than those reported by Yu et al. (9). It might be ascribed to the different method of IDR measurement by the two investigations. However the ranking order of the obtained IDR values is the same. The obtained solubility data was in agreement with those in literature as well (28).
Comparing rat Peff values with those of human showed a high regression correlation (R2=0.93, P<0.0001) for passively absorbed compounds confirming a close relationship between rat and human permeabilities (16). The fraction of dose absorbed (Fa) in human is the basis of the permeability classification. In the FDA Guidance, an active pharmaceutical ingredient is highly permeable when the fraction of dose absorbed is 90% or more. The recently revised WHO Guidelines sets a lower limit of 85%. (29-31). Yu et al. proposed to reduce the high permeability requirement for biowaiver from 90 to 85% (22, 32). On the basis of the relationship between permeability and fraction of dose absorbed (16), Peff values greater than 5.09Ã-10-5 and 4.7 Ã-10-5 cm/sec in rat and human respectively corresponds to Fa > 85, which were set as cut-off points for highly permeable drugs.
On the other hand, IDR correlates with the BCS solubility classification with 1-2 mg/min/cm2 as a class boundary. It is seen that antipyrin, ranitidine and metoprolol with IDRs of 56.79, 42.18 and 34.64 mg/cm2/min respectively have the higher values in comparison to others whereas carbamazepine and piroxicam have the lowest intrinsic dissolution rate in the series (IDR=0.035 and 0.07 mg/cm2/min respectively). As was mentioned earlier, this order is almost the same for solubility of mentioned drugs. However in the case of permeability this arrangement is not expected. The reason is that the investigated drugs belong to all four biopharmaceutical classes. That means a drug with high IDR value may belong to high or low permeability classes.
In the present study passively absorbed drugs are classified based on their intrinsic dissolution rates and human intestinal permeability values. IDR was expected to correlate more closely with in vivo dissolution dynamics of drug than solubility (9). Therefore it might be used to correct assignment of a drug to a specific BCS class. The proposed classification is presented in Fig. 1 and Fig. 2 using human and rat jejunal permeability respectively. Based on this classification, drugs are placed in four explicitly defined categories (I-IV) which are made by intersections of dashed lines drawn at the cutoff points for permeability and IDR. These classes are characterized as below:
Category I: Peff,rat > 5Ã- 10-5 (cm/sec) or Peff,human > 4.7Ã- 10-5 (cm/sec) , IDR > 2 (mg/min/cm2)
Examples of the compounds of this category include propranolol, metoprolol, verapamil and antipyrin which exhibit a high dissolution and absorption. However according to intestinal permeability estimates in rat, metoprolol is assigned in class III .
Category II: Peff,rat > 5Ã- 10-5 (cm/sec) or Peff,human > 4.7Ã- 10-5 (cm/sec) , IDR < 1 (mg/min/cm2)
Drugs like ketoprofen, naproxen, piroxicam, ibuprofen and carbamazepine are included in this category. Class II drugs have a high absorption but a low dissolution therefore absorption is limited primarily by drug dissolution in the gastrointestinal tract (26).
Category III: Peff,rat < 5Ã- 10-5 (cm/sec) or Peff,human < 4.7Ã- 10-5 (cm/sec) , IDR > 2 (mg/min/cm2)
Class III drugs, have high dissolution and low absorption. In vivo permeability is rate limiting step for drug absorption (26). Examples are atenolol, ranitidine and cimetidine.
Category IV: Peff,rat < 5Ã- 10-5 (cm/sec) or Peff,human < 4.7Ã- 10-5 (cm/sec) , IDR < 1 (mg/min/cm2)
Furosemide is an example of drugs of this category which exhibit a lot of problems for effective oral administration.
From the obtained results it is provided that the presented classification based on IDR and human intestinal permeability of drugs is in high agreement with previously introduced classification and most of the compounds are placed in correct categories they belong to (26). Although using the rat intestinal permeability values instead of human intestinal permeability, metoprolol was almost misclassified, considering non-feasibility of using human in intestinal perfusion studies, which is the major difficulty in assigning drugs to BCS classes, it may be suggested that determined intestinal permeability of drugs in rats could be used as a criterion for biopharmaceutical classification of compounds.
On the other hand, it was proposed that a biopharmaceutics drug disposition classification system (BDDCS) based on extent of drug metabolism could provide an alternative simple method to assign drugs in class I for a waiver of in vivo bioequivalence studies (33-35). According to this classification highly metabolized drugs exhibit high permeability. Therefore a drug is considered to be class I if it is highly soluble and highly metabolized. However this definition excludes drugs that have high absorption but are excreted unchanged in to bile and urine (33). Comparison of our results with BDDCS classification (â‰¥50% being defined as extensive metabolism) of drugs (35) shows high agreement (92% and 85% using human and rat intestinal permeability respectively) in classification of tested compounds (Table 1).
Another classification system namely dissolution-based classification was developed by Papadopoulou et al (36) using mean intestinal transit time (MITT), mean dissolution time (MDT) and mean absorption time (MAT). The comparison of this classification with our results is also shown in Table 1. However in dissolution-based classification propranolol and carbamazepine are classifies as class II and class IV drugs respectively which are expected to be assigned in class I and II respectively as was shown in other classifications in Table 1.
It seems that the presented classification could be used to waive in vivo bioavailability and bioequivalence studies for immediate release solid oral dosage forms which allows pharmaceutical companies to forego clinical bioequivalence studies, if their drug product meets the required specification. Moreover it may also be applied to new drug application (NDA) and abbreviated new drug application (ANDA) approvals as well as to scale-up and post approval changes in drug manufacturing. BCS classification can therefore save pharmaceutical companies a significant amount in development time and reduce costs.Â
In this study a biopharmaceutical classification system was developed based on two principle properties, intrinsic dissolution rate at pH 6.8 and rat/human intestinal permeability of passively absorbed drugs. This classification is almost in agreement with previously introduced classification system on the basis of drug's solubility and human intestinal permeability.