Mucoadhesive Polymers in Peroral Peptide Drug Delivery. II. Carbomer and Polycarbophil Are Potent Inhibitors of the Intestinal Proteolytic Enzyme Trypsin

Purpose. The evaluation of the inhibitory action of two mucoadhesive poly(acrylates), polycarbophil and carbomer, registered by the Food and Drug Administration (FDA), on the intestinal proteolytic enzyme trypsin. Methods. The effect of the polymers on trypsin activity by measuring the degradation of a trypsin specific substrate. Binding of Ca²⁺ ions and proteins (¹²⁵I-BSA) to the poly(acrylates). The influence of the polymers on the secondary trypsin structure by circular dichroism. Results. Trypsin inhibition was found to be time-dependent upon addition of Ca²⁺ in the degradation experiment. Only when Ca²⁺ was added within 10 min after trypsin incubation, recovery of the enzyme could be observed. Both polymers showed a strong Ca²⁺ binding ability. Carbomer, which had a higher inhibitory effect on trypsin activity, also revealed a higher Ca²⁺ binding affinity than polycarbophil. The amount of Ca²⁺ depleted out of the trypsin structure and the reduction of enzyme activity were comparable. Immobilization of trypsin by binding to the polymers could not be observed at pH 6.7. Circular dichroism studies suggested that, under depletion of Ca²⁺ from trypsin, the secondary structure changed its conformation, followed by an increased autodegradation of the enzyme. Conclusions. The poly(acrylates) investigated may have potential to protect peptides from tryptic degradation and may be used to master the peroral delivery of peptide drugs.     

N-Trimethylated Chitosan Chloride (TMC) Improves the Intestinal Permeation of the Peptide Drug Buserelin In Vitro (Caco-2 Cells) and In Vivo (Rats)

Purpose. To evaluate N-trimethyl chitosan chloride (TMC) of highdegrees of substitution as intestinal permeation enhancers for thepeptide drug buserelin in vitro using Caco-2 cell monolayers, and toinvestigate TMCs as enhancers of the intestinal absorption of buserelinin vivo, in rats. Methods. TMCs were tested on Caco-2 cells for their efficiency toincrease the paracellular permeability of the peptide buserelin. For thein vivo studies male Wistar rats were used and buserelin wasadministered with or without the polymers intraduodenally. Both types ofexperiments were performed at pH 7.2. Results. Transport studies with Caco-2 cell monolayers confirmed thatthe increase in buserelin permeation is dependent on the degree oftrimethylation of TMC. In agreement with the in vitro results, in vivodata revealed highly increased bioavailability of buserelin followingintraduodenal co-administration with 1.0% (w/v) TMCs.Intraduodenally applied buserelin resulted in 0.8% absolute bioavailability,whereas co-administrations with TMCs resulted in mean bioavailabilityvalues between 6 and 13 %. Chitosan HCl (1.0% pH = 7.2) did notsignificantly increase the intestinal absorption of buserelin. Conclusions. Both the in vitro and in vivo results indicate that TMCsare potent mucosal permeation enhancers of the peptide drug buserelinat neutral pH values.     

Improved Prediction of In Vivo Peroral Absorption from In Vitro Intestinal Permeability Using an Internal Standard to Control for Intra- and Inter-Rat Variability

Purpose. To evaluate the use of an in vitrointestinal permeability model to predict rat and human absorption as well as to evaluate the use of an internal standard to control for intra- and inter-rat variability. Methods. In vivoperoral absorption and in vitro steady-state intestinal permeability coefficients were determined in the rat for a variety of structurally different compounds with different physicochemical properties including: progesterone, hydrocortisone, salicylic acid, caffeine, clonidine, p-aminoclonidine, UK-14304, oxymetazoline, mannitol, PEG 900, PEG 4000, and a number of novel hydrophilic chemical entities. Results. The intestinal permeability coefficients determined in vitro could be used to predict the peroral absorption of a compound in both the rat and human. Normalizing the permeability of a test compound to an internal standard, e.g. mannitol, greatly improved the prediction of peroral absorption. Conclusions. The use of an internal standard can aid in the prediction of the peroral absorption of a test compound, in particular, for one that has moderate absorption in the range of 20–80%. Moreover, these methods would appear to be a useful means to improve the prediction of other absorption models as well, such as the Caco-2 cell systems and in-situ perfusion methods.     

Monitoring the in Vivo Delivery of Proteins from Carbomer Hydrogels by X-Ray Fluorescence

Purpose. To measure the effect of protein size on their disappearance from subcutaneously implanted carbomer hydrogel matrices. Methods. A series of different molecular weight (MW) proteins were iodinated, incorporated into Carbopol hydrogels, injected subcutaneously into rats, and monitored using X-ray fluorescence (XRF). Results. A 10 mg/mL minimum concentration of Carbopol-940 was necessary before protein [50 mg/mL iodinated bovine serum albumin (I-BSA)] retention times increased with increasing hydrogel concentration. The decreasing protein signal was not caused by outward protein diffusion or iodoprotein hydrolysis. As the protein MW increased, protein retention times lengthened [e.g., 6.2 h for insulin (5.7 kDa) to 13.3 h for thyroglobulin (669 kDa)]. Protein disappearance was monophasic first-order for some proteins and biphasic first-order for others. The disappearance rate constant ranged from 0.093 ± 0.005 h^– _1/2 to 0.187 ± 0.057 h^– _1/2, indicating gel erosion rather than protein diffusion as the rate-limiting mechanism. Entrapped I-BSA in Carbopol-1342 NF, pH 7.4, and Carbopol 2001-ETD, pH 7.4, gel matrices yielded different disappearance rates and profiles than Carbopol-940. The overall 50% disappearance rate of I-BSA was greatest for Carbopol-1342 NF (41 ± 8 h), followed by Carbopol-2001 ETD (25 ± 2 h) and Carbopol-940 (10.5 ± 0.7 h). Conclusion. XRF is a noninvasive technique that can be used to follow the status of macromolecules in vivo.     

Intestinal Absorption of Octreotide Using Trimethyl Chitosan Chloride: Studies in Pigs

Purpose. To investigate the enhancing effect of trimethyl chitosan chloride (TMC) on the enteral absorption of octreotide and to delineate the required doses of both TMC and peptide in vivo in juvenile pigs. Methods. Six female pigs (body weight, 25 kg) were operated to induce a stoma at the beginning of their jejunum and to insert an in-dwelling fistula for intrajejunal (IJ) administration of the formulations. A silicone cannula was inserted at the jugular vein for blood sampling. One week after surgery the pigs received IJ octreotide solution administrations with or without TMC at pH 7.4 or chitosan HCl at pH 5.5. For determining bioavailability (F) values, the pigs also received an octreotide solution intravenously (IV). Blood samples were taken from the cannulated jugular vein and subsequently analyzed by radioimmunoassay. Results. Intrajejunal administration of 10 mg octreotide without any polymer (control solution) resulted in F values of 1.7 ± 1.1% (mean ± SE). Chitosan HCl 1.5% (w/v) at pH 5.5 led to a 3-fold increase in F compared to the control (non-polymer containing) formulations. Co-administration of octreotide with 5 and 10% (w/v) TMC at pH 7.4 resulted in 7.7- and 14.5-fold increase of octreotide absorption, respectively (F of 13.9 ± 1.3% and 24.8 ± 1.8%). IJ administration of 5 mg octreotide solutions resulted in low F values of 0.5 ± 0.6%, whereas co-administration with 5% (w/v) TMC increased the intestinal octreotide bioavailability to 8.2 ± 1.5%. Conclusions. Cationic polymers of the chitosan type are able to enhance the intestinal absorption of the peptide drug octreotide in pigs. In this respect, TMC at neutral pH values of 7.4 appears to be more potent than chitosan HCl at a weak acidic pH of 5.5.     

In Vivo Model for Ciclosporin Intestinal Absorption in Lipid Vehicles

The influence of lipid vehicles on the intestinal absorption of Ciclosporin was studied in vivo. The model takes into account the effect of the intestinal lipid digestion on the absorption after intraduodenal administration of [³H]Ciclosporin in olive oil or middle-chain triglyceride (MCT) to the bile duct-cannulated rat. Digested vehicles significantly promoted the absorption compared to nondigested vehicles. In the nondigested state, olive oil was a significantly better vehicle than MCT, whereas the difference between both lipids was only a trend in the digested state. Further studies with variants of this in vivo model should determine the influence of abnormalities of fat digestion and absorption on the pharmacokinetics and pharmacodynamics of a drug with a low therapeutical index.     

Evaluation of Mucoadhesive Polymers in Ocular Drug Delivery. I. Viscous Solutions

The potential of a mucoadhesive polymer as an ophthalmic vehicle is evaluated within the rabbit. Precorneal clearance of a mucoadhesive polymer solution (Carbopol 934P) is compared to that of an equiviscous nonmucoadhesive poly(vinyl alcohol) solution (PVA) and buffer (PBS). The precorneal retention of the Carbopol 934P, as studied by lacrimal dacryoscintigraphy, is shown to be significantly greater (P < 0.05) than that of PVA, which, in turn, is significantly greater than that of PBS. The effect of the polymer solution on the bioavailability of pilocarpine is subsequently assessed by measuring the relative miotic response intensities produced by a 1% solution of the drug. Carbopol 934P solution produces a significant increase (P < 0.05) in bioavailability as compared to PVA and PBS. The bioavailability from PVA is significantly greater (P < 0.05) than that from PBS. Studies evaluating vehicle-drug association indicated no binding of the drug to the polymer.     

In Vitro/in Vivo Models for Peptide Oral Absorption: Comparison of Caco-2 Cell Permeability with Rat Intestinal Absorption of Renin Inhibitory Peptides

Pharmaceutical Research -     

Evaluation of Mucoadhesive Polymers in Ocular Drug Delivery. II. Polymer-Coated Vesicles

Association of Carbopol 934P and Carbopol 1342 (a hydrophobic modified Carbopol resin) with phospholipid vesicles was assessed by photon correlation spectroscopy and microelectrophoresis at pH 7.4 and 5. The precorneal clearance of the polymer-coated vesicles was compared to that of uncoated vesicles by lacrimal dacryoscintigraphy in the rabbit. The mucoadhesive polymer-coated vesicles demonstrated significantly enhanced precorneal retention compared to noncoated vesicles only at pH 5 (P < 0.005). The entrapment and subsequent release of tropicamide from Carbopol 1342-coated and uncoated liposomes were determined in vitro together with an in vivo evaluation of the vesicles formulated at the lower pH. Mucoadhesive polymer-coated vesicles failed to increase significantly the bioavailability of the entrapped tropicamide compared to uncoated vesicles and aqueous solution.     

Analysis of Drug Permeation Across Caco-2 Monolayer: Implication for Predicting In Vivo Drug Absorption

Purpose. The aim of the present work is to characterize in vitro drug permeation processes across Caco-2 monolayer and to identify the advantages of this cultured cell system in predicting in vivo drug absorption after oral administration. Methods. The passive permeability of various drugs through Caco-2 monolayer was measured using Ussing-type chambers and compared with that of the isolated rat jejunum and colon. The in vivo drug permeability to the intestinal membrane was estimated by means of an intestinal perfusion study using the rat jejunum. Results. In Caco-2 monolayer, drug permeability increased with increasing drug lipophilicity and showed a good linear relationship with the in vivo permeability. In contrast, in the isolated jejunum and colon, the permeability of high lipophilic drugs was almost constant and, propranolol, a drug with the highest lipophilicity, hardly passed through the jejunal membrane in vitro. As a result, there was no significant relationship between in vitro and in vivo drug permeability in rat jejunum. However, the amount of drugs accumulated in the jejunal mucosa increased with increasing drug lipophilicity even under the in vitro condition. Conclusions. The permeation and the accumulation studies suggested that the rate-limiting process of in vitro permeation of lipophilic drugs through the intestinal membrane differs from that of in vivo drug absorption. On the other hand, drug permeation through Caco-2 monolayer, which consists of an epithelial cell layer and a supporting filter, is essentially the same process as that of in vivo drug absorption. We concluded that the simple monolayer structure of a cultured cell system provides a distinct advantage in predicting in vivo drug absorption.     

In Vitro and in Vivo Investigations of Dihydropyridine-Based Chemical Delivery Systems for Anticonvulsants

A dihydropyridine-based chemical delivery system (CDS), intended to improve drug delivery to the brain, was investigated with a series of analogues of the anticonvulsant stiripentol. In vitro experiments demonstrated that the rates of hydrolysis of the corresponding pyridinium conjugates were influenced markedly by small changes in the structure of the drug moiety to be released. Thus, allylic esters were hydrolyzed rapidly to drug in all aqueous media, while the analogous saturated esters and an allylic amide derivative were almost totally stable. The mechanism of hydrolysis, which is particular to this series of CDS conjugates, appeared to occur via ionization to a resonance-stabilized carbocation intermediate. The same CDS compounds were investigated in vivo and compared to the corresponding drugs after intravenous administration. Only those CDS compounds that were found to hydrolyze in vitro released appreciable amounts of drug in vivo. Prolonged release of the drug from the CDS in the brain could be demonstrated for these compounds, but the gain in the ratio of brain-to-plasma AUC when the CDS was administered depended on the innate distribution characteristics of the drug. Thus, the drug D3, which had a high brain-to-plasma AUC ratio, did not show an improvement in this ratio when administered as CDS3. In contrast, stiripentol with a poor brain-to-plasma AUC ratio showed a two- to threefold increase in this ratio when administered as a CDS. These investigations highlight the need for a thorough understanding of the mechanism of drug release and the importance of the pharmacokinetic properties of the drug in designing a carrier system for delivery of drugs to the brain.     

Intestinal Drug Absorption and Metabolism in Cell Cultures: Caco-2 and Beyond

Pharmaceutical Research -     

Investigations on the Percutaneous Absorption of the Antidepressant Rolipram in Vitro and in Vivo

In vitro experiments using full-thickness human skin showed that it was feasible to deliver therapeutic amounts of the new antidepressant drug rolipram. Simple transdermal devices were constructed, and the presence of isopropyl myristate (IPM) in a silicone adhesive (Dow Corning X7-2920) enhanced the flux across excised human skin. The steady-state fluxes from adhesive mixtures containing 0, 5, and 10% IPM were 3, 5.2, and 6 µg/cm²/hr, respectively. The in vitro experiments were confirmed in a clinical study involving six healthy male volunteers. The formulations tested were an alcoholic solution and adhesive patches containing 5 and 10% IPM. The dose of drug administered was 0.5 mg/cm² and the device size 25 cm². Blood samples were withdrawn over a 24-hr period and analyzed using radioimmunoassay. The topical applications were well tolerated, with only mild or no side effects. A lag time of approximately 2 hr was found for the detection of rolipram in the plasma (detection limit, 50 pg/ml). Interindividual variations both for the peak drug levels and throughout the delivery were quite high but this magnitude of variation has been observed in many other transdermal studies. Plasma levels between 1 and 2 ng/ml were found for all formulations and the AUC_0–30hr was significantly higher for the patch containing 5% IPM.     

In Vivo Percutaneous Absorption of Hydrocortisone: Multiple-Application Dosing in Man

Percutaneous absorption of hydrocortisone was measured in six healthy adult men from whom informed consent had been obtained. The study compared a single topical dose to multiple-topical dose treatments (one vs three applications) on the same day. ¹⁴C-Labeled hydrocortisone in acetone was applied to 2.5 cm² of ventral forearm skin and protected with a nonocclusive polypropylene chamber. The amount of ¹⁴C measured in urine collected over 7 days was used to determine hydrocortisone absorption. The treatments, performed 2 to 3 weeks apart, each utilized adjacent sites on the same individuals. A single dose of 13.33 µg/cm² delivered 0.056 µg/cm² of hydro-cortisone through the skin. When the single dose was tripled to 40 µg/cm², the amount delivered through the skin increased by nearly three times, from 0.056 to 0.140 µg/cm²; the expected delivery was 3 x 0.056 µg/cm² = 0.168 µg/cm². Three serial doses of 13.33 µg/ cm² (total, 40 µg/cm²) were also expected to deliver 0.168 µg/cm² with or without soap and water washing between doses, but the observed amount of hydrocortisone delivered through the skin significantly exceeded our expectations. This indicates that multiple-dosing treatments resulted in a significant increase in bioavailability. It is postulated that increased vehicle application and washing dissolved and mobilized previously dosed hydrocortisone and increased bioavailability.     

In Vivo Properties of an In Situ Forming Gel for Parenteral Delivery of Macromolecular Drugs

Purpose. This study characterizes the in vivoproperties of an in situforming gel, comprising an IPC of water-soluble polymers, PMA and PEG, for sustained release of macromolecular drugs. Methods. 40, 50 or 60% w/v formulations were injected subcutaneously in a rat model either alone, or containing model macromolecules, 3A2-ATG-psODN or REV-psODN, to (i) determine the approximate gelling and residence time of the gel at the site of injection, (ii) assess the biological efficacy of the formulation using a MZ sleep time model, and (iii) demonstrate specificity of the sequence and selectivity of the psODNs by measuring changes in microsomal enzyme levels and urine volumes. Results. A sol to gel transition requires 15 min in vivo, and the 60% w/v IPC gel remains at the site of injection for up to 72 hr. The MZ sleep times and CYP3A2 expression due to 3A2-ATG-psODNs released from the gel are significantly different compared to that of REV-psODNs. Conclusions. The IPC solutions exhibit phase transformation in vivo, and demonstrate no evidence of toxicity. The pharmacological effects observed from the of release of 3A2-ATG-psODNs suggest that the formulation can entrap, protect, and sustain the delivery of macromolecules.     

A Theoretical Basis for a Biopharmaceutic Drug Classification: The Correlation of in Vitro Drug Product Dissolution and in Vivo Bioavailability

A biopharmaceutics drug classification scheme for correlating in vitro drug product dissolution and in vivo bioavailability is proposed based on recognizing that drug dissolution and gastrointestinal permeability are the fundamental parameters controlling rate and extent of drug absorption. This analysis uses a transport model and human permeability results for estimating in vivo drug absorption to illustrate the primary importance of solubility and permeability on drug absorption. The fundamental parameters which define oral drug absorption in humans resulting from this analysis are discussed and used as a basis for this classification scheme. These Biopharmaceutic Drug Classes are defined as: Case 1. High solubility-high permeability drugs, Case 2. Low solubility-high permeability drugs, Case 3. High solubility-low permeability drugs, and Case 4. Low solubility-low permeability drugs. Based on this classification scheme, suggestions are made for setting standards for in vitro drug dissolution testing methodology which will correlate with the in vivo process. This methodology must be based on the physiological and physical chemical properties controlling drug absorption. This analysis points out conditions under which no in vitro-in vivo correlation may be expected e.g. rapidly dissolving low permeability drugs. Furthermore, it is suggested for example that for very rapidly dissolving high solubility drugs, e.g. 85% dissolution in less than 15 minutes, a simple one point dissolution test, is all that may be needed to insure bioavailability. For slowly dissolving drugs a dissolution profile is required with multiple time points in systems which would include low pH, physiological pH, and surfactants and the in vitro conditions should mimic the in vivo processes. This classification scheme provides a basis for establishing in vitro-in vivo correlations and for estimating the absorption of drugs based on the fundamental dissolution and permeability properties of physiologic importance.     

In Silico Prediction of Optimal in Vivo Delivery Properties Using Convolution-Based Model and Clinical Trial Simulation

Purpose. To develop a new strategy for the in silico evaluation of the optimal in vivo delivery properties of a drug, minimizing a cost function defined by the brain receptor occupancy obtained in positron-emission tomography experiments. Methods. A convolution-based model was formulated to link in vivo delivery rate to plasma concentrations whereas a second-stage model was used to link plasma concentrations to the pharmacodynamic effect. A feedback control approach was applied to identify the optimal in vivo delivery rate given an appropriate optimality criterion. Finally, clinical trial simulation was used as a supportive tool for decision-making by evaluating different scenarios accounting for pharmacokinetic/pharmacodynamic parameter uncertainty, inter-subject variability, and drug potency. Results. The results revealed that the mean in vivo delivery time significantly affects brain receptor occupancy whereas the fraction of the dose available for the systemic circulation shows the highest influence on brain receptor occupancy for a givenin vivo delivery rate. Finally, variability on receptor occupancy seems to be more affected by the inter-individual variability on the disposition PK parameters. Conclusion. The integration of convolution-based model, feedback control approach, and clinical trial simulation offers a unique tool for in ilico improvement of the drug development process by identifying critical issues on drug properties, optimal in vivo delivery rate, and potential problems related to the inter-individual variability.     

Transdermal Delivery of the Synthetic Cannabinoid WIN 55,212-2: In Vitro/in Vivo Correlation

PURPOSE: The aim of the current investigation was to evaluate the percutaneous absorption of the synthetic cannabinoid WIN 55,212-2 in vitro and in vivo. METHODS: The in vitro permeation studies of WIN 55,212-2 in human skin, hairless guinea pig skin, a polymer membrane with adhesive, and a skin/polymer membrane composite were conducted in flowthrough diffusion cells. The pharmacokinetic parameters for WIN 55,212-2 were determined after intravenous administration and topical application of Hill Top Chambers and transdermal therapeutic systems (TTS) in guinea pigs. RESULTS: The in vitro permeation studies indicated that the flux of WIN 55,212-2 through hairless guinea pig skin was 1.2 times more than that through human skin. The flux of WIN 55,212-2 through human and guinea pig skin was not significantly higher than that through the corresponding skin/polymer membrane composites. The mean guinea pig steady-state plasma concentrations after topical 6.3 cm2 chamber and 14.5 cm2 TTS patch applications were 5.0 ng/ml and 8.6 ng/ml, respectively. CONCLUSIONS: The topical drug treatments provided significant steady-state plasma drug levels for 48 h. The observed in vivo results from the Hill Top Chambers and TTS patches in the guinea pigs were in good agreement with the predicted plasma concentrations from the in vitro data.     

Release and Absorption Characteristics of Novel Theophylline Sustained-Release Formulations: In Vitro-in Vivo Correlation

Five new experimental sustained-release (SR) formulations of theophylline, T-l, T-l-A, T-2, T-2-A, and T-2-E, in a matrix tablet form with a protein were developed. The in vitro release of theophylline from these novel experimental formulations and two commercial (Theotrim and Theo-Dur) SR formulations, was studied for 2 hr immersed in simulated gastric fluid TS, followed by an additional 10 hr immersed in simulated intestinal fluid TS. Like Theotrim and Theo-Dur, theophylline release profiles from all the novel experimental formulations were smooth, controlled, and unaffected by changes in the pH and the proteolytic enzyme content of the incubation media. Pharmacokinetic evaluation of T-l, T-l-A, T-2-A, Theotrim, and Theo-Dur was carried out in five dogs and six healthy human volunteers under fasting conditions, using immediate-release aminophylline tablets as controls. Pharmacokinetic analysis by the Wagner–Nelson procedure revealed sustained-release absorption characteristics for all the formulations with the exception of the immediate release aminophylline tablet. For each of the formulations tested, the regression analysis results of the percentage of theophylline absorbed in dogs or humans against the mean percentage released in vitro, at the corresponding times, indicated a high correlation. These data imply that the in vivo release profiles under fasting conditions in the gastrointestinal tract of dogs and humans may be similar to those in the in vitro studies.     

Site-Specific Drug Delivery to Pilosebaceous Structures Using Polymeric Microspheres

In order to improve the therapeutic index of adapalene, a new drug under development for the treatment of acne, site-specific delivery to the hair follicles using 50:50 poly(DL-lactic-co-glycolic acid) microspheres as particulate carriers was investigated in vitro and in vivo. The percutaneous penetration pathway of the microspheres was shown to be dependent on their mean diameter. Thus, after topical application onto hairless rat or human skin, adapalene-loaded microspheres (5-µm diameter) were specifically targeted to the follicular ducts and did not penetrate via the stratum corneum. The in vitro release of adapalene from the microspheres into artificial sebum at 37°C was controlled and faster than the in vivo sebum excretion in humans. Aiming to reduce either the applied dose of drug or the frequency of administration, different formulations of adapalene-loaded microspheres were evaluated in vivo in the rhino mouse model. A dose-related comedolytic activity of topical formulations of adapalene-loaded microspheres was observed in this model. Furthermore, by applying a site-specific drug delivery system (0.1% adapalene) every other day or by administering a 10-fold less concentrated targeted formulation (0.01%) every day, a pharmacological activity equivalent to a daily application of an aqueous gel containing drug crystals (0.1% adapalene) was observed. Since an aqueous gel containing 10% adapalene-loaded microspheres was not irritating in a rabbit skin irritancy test, this formulation was applied onto forearms of human volunteers. Site-specific drug delivery was further evidenced by follicular biopsy. These results support the view that follicular drug targeting using 5-µm polymeric microspheres may represent a promising therapeutic approach for the treatment of pathologies associated with pilosebaceous units.