Mean Residence Time Of Drug Research Articles

Transdermal delivery of doxorubicin and methotrexate from polyelectrolyte three layer nanoparticle of graphene oxide/polyethyleneimine/dextran sulphate for chemotherapy: In vitro and in vivo studies

In the present work, novel dual drug loaded - anionic graphene oxide/cationic polyethyleneimine/poly anionic dextran sulfate (GO/PEI/DS) was synthesized by a layer-by-layer self-assembly technique for transdermal anti-cancer drug delivery. Doxorubicin (DOX) was loaded onto folic acid conjugated graphene oxide (GO) and methotrexate (MTX) was loaded onto dextran sulfate (DS). The drug carrier was characterized by fourier transform infrared spectroscopy, x-ray diffraction spectroscopy, dynamic light scattering analysis, scanning electron microscopy, atomic force microscopy, transmission electron microscopy, zeta-potential analysis, drug loading and encapsulation efficiency, equilibrium swelling studies, in vitro drug release studies, hemocompatibility assays, in vitro cytotoxicity assay, skin irritation studies, ex vivo skin permeation studies and in vivo pharmacokinetic studies.The cytotoxicity of synthesized materials was evaluated against MCF-7 breast cancer cells using an MTT assay and apoptosis assay. The synergistic effect of DOX and MTX to inhibit the growth of cancer cells was also confirmed. The final dual drug-loaded carrier showed a cell viability of 36.38 % towards MCF-7 cancer cells while it showed a cell viability of >87.65 % towards normal L929 cell lines. The in vivo pharmacokinetic studies in Wistar rats through oral and transdermal routes of administration, the transdermal route of administration of dual drug-loaded material provided a sustained and prolonged drug existence in the blood circulation system. The mean residence time of drugs in the bloodstream via oral administration was 83.98 ± 3.71 h, while that of the transdermal route of administration was 149.62 ± 6.11 h. The results revealed that the synthesized dual drug-loaded material showed a good pH-dependent controlled and sustained release profile for both DOX and MTX via the transdermal route of administration in comparison with oral delivery.

Enhanced oral bioavailability and neuroprotective effect of fisetin through its SNEDDS against rotenone-induced Parkinson's disease rat model

Fisetin (FS) was reported to have various pharmacological activities. But due to its lower aqueous solubility and oral bioavailability, it is not in much use. As solubility and bioavailability plays and important role in the pharmacological activity, in this research work we tried to improve the oral bioavailability of fisetin. In this research work, we developed self-nanoemulsifying drug delivery system (SNEDDS) of fisetin. Developed SNEDDS were subjected for pharmacokinetic and pharmacodynamics studies against rotenone-induced Parkinson's disease (PD) model in rats. Higher Cmax and area under the curve during pharmacokinetic study indicated that SNEDDS improved the oral bioavailability of FS and also increased the mean residence time of drug in plasma. Results of behavior parameters (locomotor, muscle co-ordination and catalepsy), biochemical estimation (TBARS, nitrite, GSH, SOD and CAT) and ELISA (soluble alfa synuclein, BDNF, TNF-α and IL-6) confirmed the significantly improved (p < 0.05) neuroprotection in rats treated with FS loaded SNEDDS as compared to rats treated with naïve FS. This study suggests that SNEDDS improved the oral bioavailability of FS which further helped in improving its neuroprotective activity in rat model of PD. It further suggests the potential use of FS-SNEDDS in effective management of PD condition.

Development of micropore-controlled release tablet and in vivo pharmacokinetic study

The aim of this work was to develop a micropore-controlled release tablet for theophylline. The tablets were composed of a drug core surrounded by a microporous film. The major components of coating film included a biocompatible semipermeable polymer, cellulose acetate, and a water-soluble pore-forming agent, polyethylene glycol). The effect of the coating film composition and the type of excipient incorporated in the drug core on drug release were demonstrated via an in vitro release study. The optimized formulation was further investigated in vivo of rabbits. The results showed that micropore-controlled release tablets continuously released drug for 24-36 hours depending on the type of excipient in the drug core and the coating film composition. Incorporation of lactose in the drug core enhanced drug release from micropore-controlled release tablets. In vivo animal study revealed that the micropore-controlled release tablets reduced the maximum concentration and prolonged the mean residence time of drug.

Nanostructured lipid carriers for transdermal delivery of acid labile lansoprazole

The aim of this study was to develop nanostructured lipid carriers (NLCs) for transdermal delivery of acid-labile lansoprazole (LPZ). The drug loading, particle size, zeta potential, thermal behavior and stability of NLCs were evaluated. The particle size of NLCs was in the range of 90−210nm and the zeta potential was −61.9 to +3.2mV dependent of the compositions. Stearylamine (SA) prevented lansoprazole degradation and maintained drug stable in NLCs. The anionic sodium dodecyl sulfate (SDS) adsorbed on the lipid surface and formed complex with cationic SA to prevent NLCs aggregation. The effects of type (e.g., isopropyl myristate (IPM), menthol) and concentration (e.g., 1.25, 2.50, 3.75%w/w) of enhancers on penetration of lansoprazole NLC hydrogels were investigated in vitro using Wistar rat skin. The steady-state flux of lansoprazole NLC hydrogel containing 3.75% IPM was the highest which was enhanced by 2.7 folds as compared to enhancer-free NLC hydrogel. In vivo pharmacokinetics of lansoprazole following transdermal delivery of NLC hydrogel showed that the elimination of drug was significantly reduced and the mean residence time of drug was prominently prolonged as compared to intravenous drug solution (p<0.005). The accumulation of drug in the skin and continuous penetration of drug through the skin accounted for the maintenance of drug concentration for at least 24h.

On the accuracy of calculation of the mean residence time of drug in the body and its volumes of distribution based on the assumption of central elimination

1. The steady state and terminal volumes of distribution, as well as the mean residence time of drug in the body (Vss, Vβ, and MRT) are the common pharmacokinetic parameters calculated using the drug plasma concentration–time profile (Cp(t)) following intravenous (iv bolus or constant rate infusion) drug administration.2. These traditional calculations are valid for the linear pharmacokinetic system with central elimination (i.e. elimination rate being proportional to drug concentration in plasma). The assumption of central elimination is not valid in general, so that the accuracy of the traditional calculation of these parameters is uncertain.3. The comparison of Vss, Vβ, and MRT calculated by the derived exact equations and by the commonly used ones was made considering a physiological model. It turned out that the difference between the exact and simplified calculations does not exceed 2%.4. Thus the calculations of Vss, Vβ, and MRT, which are based on the assumption of central elimination, may be considered as quite accurate. Consequently it can be used as the standard for comparisons with kinetic and in silico models.

Positively Charged Polymeric Nanoparticle Reservoirs of Terbinafine Hydrochloride: Preclinical Implications for Controlled Drug Delivery in the Aqueous Humor of Rabbits

Frequent instillation of terbinafine hydrochloride (T HCl) eye drops (0.25%, w/v) is necessary to maintain effective aqueous humor concentrations for treatment of fungal keratitis. The current approach aimed at developing potential positively charged controlled-release polymeric nanoparticles (NPs) of T HCl. The estimation of the drug pharmacokinetics in the aqueous humor following ocular instillation of the best-achieved NPs in rabbits was another goal. Eighteen drug-loaded (0.50%, w/v) formulae were fabricated by the nanopreciptation method using Eudragit® RS100 and chitosan (0.25%, 0.5%, and 1%, w/v). Soybean lecithin (1%, w/v) and Pluronic® F68 (0.5%, 1%, and 1.5%, w/v) were incorporated in the alcoholic and aqueous phases, respectively. The NPs were evaluated for particle size, zeta potential, entrapment efficiency percentage (EE%), morphological examination, drug release in simulated tear fluid (pH 7.4), Fourier-transform IR (FT-IR), X-ray diffraction (XRD), physical stability (2 months, 4°C and 25°C), and drug pharmacokinetics in the rabbit aqueous humor relative to an oily drug solution. Spherical, discrete NPs were successfully developed with mean particle size and zeta potential ranging from 73.29 to 320.15 nm and +20.51 to +40.32 mV, respectively. Higher EE% were achieved with Eudragit® RS100-based NPs. The duration of drug release was extended to more than 8 h. FT-IR and XRD revealed compatibility between inactive formulation ingredients and T HCl and permanence of the latter's crystallinity, respectively. The NPs were physically stable, for at least 2 months, when refrigerated. F5-NP suspension significantly (P<0.05) increased drug mean residence time and improved its ocular bioavailability; 1.657-fold.

Application of organogels as oral controlled release formulations of hydrophilic drugs

We previously demonstrated that organogels prepared from soybean oil using 12-hydroxy stearic acid as a gelator can slowly release ibuprofen, a model lipophilic drug. In this study, we investigated the applicability of organogels as controlled release formulations of hydrophilic drugs. The release rates of theophylline and ofloxacin, which are used as model hydrophilic drugs, were significantly slower than those of ibuprofen and antipyrine (model lipophilic drugs). Furthermore, no erosion was noted during drug release from organogels. Lipophilic drug molecules are released after diffusion in organogels because all molecules fully dissolve in the gel. On the other hand, hydrophilic drug molecules need to be dissolved before they diffuse in the organogel, prior to their release from the gel. Therefore, it is speculated that the release rates of hydrophilic drugs are slower than those of lipophilic drugs. To confirm the usefulness of organogels in controlled release formulations in vivo, organogels containing ibuprofen, ofloxacin, theophylline or antipyrine were intraduodenally administered to rats. All drugs used in this study were rapidly absorbed when administered in aqueous suspensions. In contrast, the drug concentrations in plasma after administration in organogels were lower; however, the lower concentrations of drugs sustained for 10 h after administration. With organogel administration, the mean residence time of drugs was longer than that with aqueous suspension administration. In conclusion, organogels are potential candidates for controlled release formulations of not only lipophilic drugs, but also hydrophilic drugs.

A Small Molecule Inhibitor Targeting CREB and CBP Inhibits Proliferation of AML Cells In Vitro and In Vivo,

Abstract 3607The cAMP Response Element Binding Protein, CREB, is a nuclear transcription factor that is a downstream target of signaling pathways regulating memory, glucose homeostasis, cell proliferation, differentiation, and survival. We previously demonstrated that CREB is overexpressed at both the protein and mRNA levels in leukemia blasts and in leukemia stem cells. AML patients who overexpress CREB in their bone marrow have an increased risk of relapse and decreased event-free survival. To determine whether CREB is sufficient for leukemogenesis, we created a transgenic mouse in which CREB is expressed under the control of a myeloid specific hMRP8 promoter. Bone marrow progenitors from CREB transgenic mice had higher proliferative potential and replating ability. These mice developed myeloproliferative disease after one year but not acute leukemia, suggesting that CREB is not sufficient to induce myeloid transformation. To determine whether CREB is necessary for leukemia cell proliferation, we transduced AML cells with lentiviral CREB shRNA. We observed that downregulation of CREB led to decreased AML cell proliferation and survival in vitro. Furthermore, our results demonstrated that CREB knockdown inhibits the growth of AML cells in vivo without affecting normal hematopoietic stem cell function. Together, these results strongly suggest that CREB acts as a proto-oncogene and is a potential target for AML therapy.CREB is activated through phosphorylation, leading to the recruitment of the histone acetyltransferase, CREB binding protein (CBP) and subsequent target gene expression. XX-650-23 (MW: 288.3) is a small molecule that was identified through in silico screening methods to inhibit the interaction between CREB and CBP. We tested the effects of the drug on various AML cell lines using MTT assays and trypan blue exclusion. The IC50 ranged from 700 nM to 2 μM after 72 hours of drug treatment. However, treatment of normal human bone marrow progenitors cultured in methylcellulose containing XX-650-23 at a concentration of 10 μM had no effect on colony numbers. We also tested the in vivo effects of XX-650-23 using xenograft NOD-SCID IL-2Rgamma null (NSG) mouse models. To assess toxicity, mice were treated with the drug at various concentrations, ranging from 10 to 20 mg/kg, by intraperitoneal injection (IP) once daily for 28 days. We did not observe any weight loss or hematologic, renal, hepatic, or cardiac toxicity in the mice. We also performed pharmacokinetic analysis to determine the half-life and stability of the drug. After 1 hour of treatment, the serum drug concentration was 33 nM. The estimated drug mean residence time was 7.5 hours. Plasma clearance divided by IP absorption fraction was 9.6 L/min/kg. The mice were treated daily with drug (17.5mg/kg IP) or vehicle control once MV4-11 cells reached a tumor size of 300mm3 or at the time cells were injected. Our results demonstrated significant inhibition of tumor growth with treatment of the drug started on the day of injection of cells compared to waiting until the tumor reached 300mm3. Together, these data suggest that a small molecule inhibitor targeting CREB and CBP interaction is a potential avenue for drug development. We are currently studying the mechanisms by which XX-650-23 inhibits AML cell proliferation and analyzing the effects of combining the drug with chemotherapy. Disclosures:No relevant conflicts of interest to declare.

The influence of hepatic transport on the distribution volumes and mean residence time of drug in the body and the accuracy of estimating these parameters by the traditional pharmacokinetic calculations

The influence of hepatic uptake and efflux, which includes passive diffusion and transporter-mediated component, on drug distribution volumes [steady-state volume of distribution (V(ss)) and terminal volume of distribution (V(β))], mean residence time (MRT), clearance, and terminal half-life is considered using a simplified physiologically based pharmacokinetic model. To account for hepatic uptake, liver is treated as two-compartmental unit with drug transfer from extracellular water into hepatocytes. The exactly calculated distribution volumes and MRT are compared with that obtained by the traditional equations based on the assumption of central elimination. It was found that V(ss) may increase more than 10-fold and V(β) more than 100-fold due to the contribution of transporter-mediated uptake. The terminal half-life may be substantially shortened (more than 100-fold) due to transporters. It may also decrease significantly due to the increase of intrinsic hepatic clearance (CL(int)), whereas hepatic clearance has already reached saturation (and stays close to the possible maximum value). It is shown that in case of transporter-mediated uptake of compound into hepatocytes, in the absence of efflux and passive diffusion (unidirectional uptake), hepatic clearance is independent of CL(int) and is determined by hepatic blood flow and uptake rate constant. The effects of transporter-mediated uptake are mostly pronounced for hydrophilic acidic compounds and moderately lipophilic neutral compounds. For basic compounds and lipophilic neutral compounds the change of distribution volumes due to transporters is rather unlikely. It was found that the traditional equations provide very accurate values of V(ss), V(β), and MRT in the absence of transporter action even for very low rates of passive diffusion. On the other hand, the traditional equations fail to provide the correct values of these parameters when the increase of distribution volumes due to transporters takes place, and actually yield the values substantially smaller than the true ones (up to an order of magnitude for V(ss) and MRT, and three orders of magnitude for V(β)).

On the Accuracy of Estimation of Basic Pharmacokinetic Parameters by the Traditional Noncompartmental Equations and the Prediction of the Steady-State Volume of Distribution in Obese Patients Based Upon Data Derived from Normal Subjects

The steady-state and terminal volumes of distribution, as well as the mean residence time of drug in the body (V(ss), V(β), and MRT) are the common pharmacokinetic parameters calculated using the drug plasma concentration-time profile C(p) (t) following intravenous (i.v. bolus or constant rate infusion) drug administration. These calculations are valid for the linear pharmacokinetic system with central elimination (i.e., elimination rate being proportional to drug concentration in plasma). Formally, the assumption of central elimination is not normally met because the rate of drug elimination is proportional to the unbound drug concentration at elimination site, although equilibration between systemic circulation and the site of clearance for majority of small molecule drugs is fast. Thus, the assumption of central elimination is practically quite adequate. It appears reasonable to estimate the extent of possible errors in determination of these pharmacokinetic parameters due to the absence of central elimination. The comparison of V(ss), V(β), and MRT calculated by exact equations and the commonly used ones was made considering a simplified physiologically based pharmacokinetic model. It was found that if the drug plasma concentration profile is detected accurately, determination of drug distribution volumes and MRT using the traditional noncompartmental calculations of these parameters from C(p) (t) yields the values very close to that obtained from exact equations. Though in practice, the accurate measurement of C(p) (t), especially its terminal phase, may not always be possible. This is particularly applicable for obtaining the distribution volumes of lipophilic compounds in obese subjects, when the possibility of late terminal phase at low drug concentration is quite likely, specifically for compounds with high clearance. An accurate determination of V(ss) is much needed in clinical practice because it is critical for the proper selection of drug treatment regimen. For that reason, we developed a convenient method for calculation of V(ss) in obese (or underweight) subjects. It is based on using the V(ss) values obtained from pharmacokinetic studies in normal subjects and the physicochemical properties of drug molecule. A simple criterion that determines either the increase or decrease of V(ss) (per unit body weight) due to obesity is obtained. The accurate determination of adipose tissue-plasma partition coefficient is crucial for the practical application of suggested method.

Intraocular Pharmacokinetics of a Crystalline Lipid Prodrug, Octadecyloxyethyl-Cyclic-Cidofovir, for Cytomegalovirus Retinitis

To evaluate the intraocular pharmacokinetics of octadecyloxyethyl-cyclic-cidofovir (ODE-cCDV) after intravitreal injection into rabbit eyes. Twenty-seven New Zealand red rabbits (27 eyes) received intravitreal injections of (14)C-labeled ODE-cCDV (100 μg drug suspended in 5% dextrose), and ocular tissues were collected from 3 rabbits at each predetermined time point (1 h, 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 5 weeks, and 9 weeks) after the injection. The eye globes were enucleated, and the vitreous, retina, and choroids were separated and harvested into pre-weighed scintillation vials. Levels of ODE-cCDV were measured by counting in a liquid scintillation counter, and pharmacokinetic (PK) parameters were determined. In addition, 3 eyes of 3 animals were used for autoradiography study at day 1, week 3, and week 6. ODE-cCDV in vitreous as a whole followed a 2-phase first-order elimination, whereas ODE-cCDV in retina and choroid manifested a nearly steady state during the first 3 weeks and then followed a first-order elimination with the apparent elimination half-life of 10.1 and 7.2 days. For vitreous, apparent elimination half-life was 25 days. However, the drug mean residence time was much longer in retina (17.6 days) and choroid (19.6 days) than that in the vitreous (11.6 days). The drug exposure to the retina [area under the curve (AUC) = 1120837.1 ng · day/mL] was greater than the exposure to the vitreous (AUC = 958645.8 ng · day/mL) and the choroid (AUC = 415407.47). A crystalline lipid prodrug, ODE-cCDV, has longer vitreous half-life than that in other ocular tissues due to its solid drug depot formation in vitreous. Over time, dissolved free ODE-cCDV from drug depot feeds and accumulates in the retina.

Inhibitor of the CREB Proto-Oncogene Suppresses AML Cell Proliferation and Induces Apoptosis In Vitro and In Vivo

AbstractAbstract 3270The cAMP Response Element Binding Protein, CREB, is a nuclear transcription factor that is critical for cell proliferation, differentiation, and survival in a variety of cell types. We previously demonstrated that CREB is overexpressed in leukemia blasts from greater than 60% of patients with AML. CREB overexpression is also associated with an increased risk of relapse and decreased event free survival in a cohort of AML patients. To understand the role of CREB in leukemogenesis, a transgenic mouse was created by overexpressing CREB under the control of the hMRP8 promoter in Gr-1/Mac-1 + myeloid cells. Bone marrow progenitors from these mice have increased proliferative potential and replating ability. CREB transgenic mice developed myeloproliferative disease after one year, but not acute myeloid leukemia suggesting that CREB is not sufficient to induce complete transformation. Given these results, we hypothesized that CREB could be a potential target for AML therapy. To test this possibility, we knocked down CREB using lentiviral shRNAs in primary mouse hematopoietic stem cells in vitro and in vivo. Downregulation of CREB resulted in significant decrease in myeloid colony numbers in methylcellulose colony assays. However, there was no effect of CREB knockdown in hematopoietic stem cell numbers or long-term engraftment by stem cell transduction and transplantation experiments. In contrast, inhibition of CREB by shRNAs in human myeloid leukemia cells and Ba/F3 cells transduced with T315I mutant BCR-ABL resulted in suppression of growth of leukemia cells in vitro and in NOD-SCID mice. Taken together, these data suggest that CREB preferentially inhibits the growth of leukemia cells without significantly affecting normal hematopoietic stem cell proliferation or function. To target CREB in AML cells, a small molecule, XX-650-23 (MW: 288.3), was developed to inhibit the interaction between CREB and its binding partner, the histone acetyltransferase CBP (CREB Binding Protein). We tested the effects of the drug on AML cell lines Molm-13 and MV-411 in vitro using MTT assays and trypan blue exclusion. The IC50 for both cell lines was between 1–2 micromolar concentration after 72 hours of treatment. Our results also demonstrated that treatment of MV-411 cells with XX-650-23 not only inhibited cell proliferation but also resulted in apoptosis. Treatment of normal human bone marrow progenitors with XX-650-23 had no effect on colony numbers in methylcellulose containing IL-3, IL-6, and Stem Cell Factor up to 10 micromolar concentration. NOD-SCID IL-2Rgamma null (NSG) mice treated with the drug at 10, 15, 17.5, and 20 mg/kg IP once daily for 28 days had no evidence of weight loss or toxicity to organs as measured by peripheral blood counts and chemistries to assess kidney and liver function. We next tested the drug in xenograft models of AML with MV-411 cells (2×106) injected subcutaneously into the flanks of NSG mice. Three groups of 5 mice were treated with either XX-650-23 or vehicle control at 17.5 mg/kg IP daily at the time of injection of MV-411 cells or after the tumors reached a size of 300 mm3. Serum drug concentration 1 hour after first injection was 33 nM. Estimated drug mean residence time was 7.5 hours, and plasma clearance was 9.6 ml/min/kg, assuming complete IP absorption. Our results demonstrated a significant inhibition of tumor growth with treatment of the drug and MV-411 cells at the same time on Day 1. There was also an effect of the drug alone when treated after the tumor reached a size of 300 mm3, although less dramatic. These data suggest that development of a drug that targets CREB is a novel approach to treat AML. We are currently optimizing the treatment of AML cells with XX-650-23 in mice and investigating the downstream signaling pathways to understand the mechanism by which XX-650-23 suppresses proliferation and induces apoptosis of AML cells. Disclosures:Sakamoto:Abbott Laboratories, Inc.: Research Funding; Genentech, Inc.: Research Funding.

Prolonged absorption of antimony(V) by the oral route from non‐inclusion meglumine antimoniate–β‐cyclodextrin conjugates

The orally active composition comprising meglumine antimoniate (MA) and beta-cyclodextrin (beta-CD) differs markedly from conventional drug-CD complexes, since it combines a water-soluble drug and a hydrophilic CD. In order to obtain insights into the mechanism(s) responsible for the improved oral delivery of the drug, physicochemical and pharmacokinetic studies were carried out. The composition investigated here was prepared at a 7:1 antimony(Sb)/beta-CD molar ratio, a condition that improves its solubility in water and allows the oral administration of a high dose of Sb in large animals. It was characterized by circular dichroism, (1)H-NMR, ESI-MS and photon correlation spectroscopy. Pharmacokinetic data were obtained in Beagle dogs after oral administration of the composition at 100 mg Sb/kg. (1)H-NMR and ESI-MS data supported the formation of non-inclusion complexes between MA and beta-CD. Sub-micron assemblies were also evidenced that slowly dissociate and presumably release the MA drug, upon reconstitution of the composition in water. Pharmacokinetic studies of MA and MA/beta-CD in dogs showed a prolongation of the serum mean residence time of Sb from 4.1 to 6.8 h, upon complexation of MA with beta-CD. Evidence was also obtained that Sb remains essentially under the form of pentavalent Sb-meglumine complex, following gastro-intestinal absorption from the MA/beta-CD composition. In conclusion, the present data support the model that the sustained drug release property of 7:1 MA/beta-CD composition resulted in the prolongation of MA absorption by the oral route and, consequently, in the increase of the drug mean residence time in serum.

Determination of Mean Residence Time of Drug in Plasma and the Influence of the Initial Drug Elimination and Distribution on the Calculation of Pharmacokinetic Parameters

The equation for the calculation of mean residence time of drug in plasma, tp, is obtained. It is shown that the previously suggested calculation of tp considerably overestimates the true value in most cases. It is suggested that due to the possible initial (before establishing the uniform drug mixing in plasma) fast elimination of drug, the commonly calculated total body clearance (Cl = D/AUC) may substantially overestimate the clearance in the linear range of elimination of well‐stirred drug. This would result in the high in vivo Cl values that are not supported by the in vitro studies of drug metabolism and stability in tissues. It is shown that the mean residence time of drug in the body, volumes of distribution, oral bioavailability and distribution clearance estimated with the account of initial drug distribution and elimination, may substantially deviate from the values obtained by the traditional calculations. © 2008 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:748–762, 2009

The basics of preclinical drug development for neurodegenerative disease indications

Preclinical development encompasses the activities that link drug discovery in the laboratory to initiation of human clinical trials. Preclinical studies can be designed to identify a lead candidate from several hits; develop the best procedure for new drug scale-up; select the best formulation; determine the route, frequency, and duration of exposure; and ultimately support the intended clinical trial design. The details of each preclinical development package can vary, but all have some common features. Rodent and nonrodent mammalian models are used to delineate the pharmacokinetic profile and general safety, as well as to identify toxicity patterns. One or more species may be used to determine the drug's mean residence time in the body, which depends on inherent absorption, distribution, metabolism, and excretion properties. For drugs intended to treat Alzheimer's disease or other brain-targeted diseases, the ability of a drug to cross the blood brain barrier may be a key issue. Toxicology and safety studies identify potential target organs for adverse effects and define the Therapeutic Index to set the initial starting doses in clinical trials. Pivotal preclinical safety studies generally require regulatory oversight as defined by US Food and Drug Administration (FDA) Good Laboratory Practices and international guidelines, including the International Conference on Harmonisation. Concurrent preclinical development activities include developing the Clinical Plan and preparing the new drug product, including the associated documentation to meet stringent FDA Good Manufacturing Practices regulatory guidelines. A wide range of commercial and government contract options are available for investigators seeking to advance their candidate(s). Government programs such as the Small Business Innovative Research and Small Business Technology Transfer grants and the National Institutes of Health Rapid Access to Interventional Development Pilot Program provide funding and services to assist applicants in preparing the preclinical programs and documentation for their drugs. Increasingly, private foundations are also funding preclinical work. Close interaction with the FDA, including a meeting to prepare for submission of an Investigational New Drug application, is critical to ensure that the preclinical development package properly supports the planned phase I clinical trial.

Making the most of sparse clinical data by using a predictive-model-based analysis, illustrated with a stavudine pharmacokinetic study

A small-scale clinical investigation was done to quantify the penetration of stavudine (D4T) into cerebrospinal fluid (CSF). A model-based analysis estimates the steady-state ratio of AUCs of CSF and plasma concentrations ( R AUC) to be 0.270, and the mean residence time of drug in the CSF to be 7.04 h. The analysis illustrates the advantages of a causal (scientific, predictive) model-based approach to analysis over a noncausal (empirical, descriptive) approach when the data, as here, demonstrate certain problematic features commonly encountered in clinical data, namely (i) few subjects, (ii) sparse sampling, (iii) repeated measures, (iv) imbalance, and (v) individual design variation. These features generally require special attention in data analysis. The causal-model-based analysis deals with features (i) and (ii), both of which reduce efficiency, by combining data from different studies and adding subject-matter prior information. It deals with features (iii)–(v), all of which prevent ‘averaging’ individual data points directly, first, by adjusting in the model for interindividual data differences due to design differences, secondly, by explicitly differentiating between interpatient, interoccasion, and measurement error variation, and lastly, by defining a scientifically meaningful estimand ( R AUC) that is independent of design.

Volumes of distribution and mean residence time of drugs with linear tissue distribution and binding and nonlinear protein binding.

Based on a generalized model, equations for calculating the mean residence time in the body at single dose (MRT) and at steady state (MRTss), apparent steady-state volume of distribution (Vss), and steady-state volume of distribution (Vss) are derived for a drug exhibiting nonlinear protein binding. Interrelationships between Vss and Vss as well as between MRT and MRTss are also discussed and illustrated with simulated data. In addition, a method for estimating the central volume of distribution of the bound drug and the sum of the central volume of distribution of the unbound drug and the area under the first moment curve of distribution function for drugs with nonlinear protein binding is proposed and illustrated with both simulated and published data.

Mean residence time of drugs administered non‐instantaneously and undergoing linear tissue distribution and reversible metabolism and linear or non‐linear elimination from the central compartment

Based on disposition decomposition analysis (DDA), equations for the mean residence times (MRT) in the body are derived for a drug and its interconversion metabolite that undergo linear tissue distribution and linear or non-linear elimination from the central compartment after non-instantaneous administration of the drug. The MRT of the drug after non-instantaneous input can be related to the MRT of the drug after intravenous administration, the ratio of the total area under the plasma concentration-time curve of the drug after non-instantaneous administration to that after intravenous administration, the bioavailability of the drug, and the mean input time of the drug. Similar relationships also exist for the MRT of the interconversion metabolite after non-instantaneous input of the drug. The application of these equations to a non-linear reversible metabolic system is illustrated with computer simulations.

Mean residence time of oral drugs undergoing first-pass and linear reversible metabolism.

Equations for the mean residence times in the body (MRT) and AUMC/AUC of a drug and its metabolite have been derived for an oral drug undergoing first-pass and linear reversible metabolism. The mean residence times of the drug or interconversion metabolite in the body after oral drug are described by equations which include the mean absorption time (MAT), the mean residence times of the drug or metabolite in the body after intravenous administration of the drug, the fractions of the dose entering the systemic circulation as the parent drug and metabolite, and the systemically available fractions of the drug (Fpp) or metabolite (Fpm). Similarly, the AUMC/AUC of the drug and metabolite after oral drug can be related to the MAT, ratios of the fraction of the dose entering the systemic circulation to the systemically available fraction, the first-time fractional conversion of each compound, and AUMC/AUC ratios after separate intravenous administration of each compound. The Fpp and Fpm values, in turn, are related to the first-pass availabilities of both drug and metabolite and the first-time fractional conversion fractions. The application of these equations to a dual reversible two-compartment model is illustrated by computer simulations.

Erythrocytes as a Total Barrier for Renal Excretion of Hydrochlorothiazide: Slow Influx and Efflux across Erythrocyte Membranes

The potential barrier effect of erythrocytes (RBC) on renal excretion (mainly by tubular secretion) of hydrochlorothiazide (HCTZ) was evaluated in nine anesthetized rats during steady-state iv infusion. Drug concentrations in plasma and blood from the carotid artery and renal vein were assayed by a simple modified HPLC method. Renal extraction ratios were concentration-independent with a mean of 0.17 ± 0.05 (SD). The renal excretion was found to occur primarily from the drug in plasma; the mean net fractional removal from plasma was 0.57 ±0.12, while that from RBC was <0.008 ± 0.041. The virtual total unavailability of HCTZ from RBC (containing ~70% of drug in arterial blood) for renal excretion is attributed to relatively slow efflux of drug from RBC to plasma during each passage through the kidney compared with the blood transit time (in seconds). Preliminary in vitro influx and efflux kinetics of HCTZ across RBC membranes were studied using rat and human blood. The flux data could be adequately described by a linear, reversible, closed two-component system model, and the mean equilibration half-times (ET1/2) in rat and human blood were 10.9 and 20.5 min, respectively. The mean residence time of drug in blood circulation of rats was estimated to be 8.32 ± 1.06 min, which is shorter than the ET1/2. This is consistent with data indicating that distribution equilibrium of HCTZ in arterial blood might not be reached in vivo even at steady state. Other implications of slow transport kinetics of drugs across RBC membranes are discussed.