Challenges, approaches and enablers: effectively triangulating towards dose selection in pediatric rare diseases.

Dose selection is an integral part of a molecule's journey to become medicine. On top of typical challenges faced in dose selection for more common diseases, pediatric rare disease has additional unique challenges due to the combination of 'rare' and 'pediatric' populations. Using the central theme of maximizing 'relevant' information to overcome information paucity, dose selection strategy in pediatric rare diseases is discussed using a triangulation concept involving challenges, approaches and very importantly, enablers. Using actual examples, unique scenarios are discussed where specific enablers allowed certain approaches to be used to overcome the challenges. The continued need for model-informed drug development is also discussed using examples of where modeling and simulation tools have been successfully used in bridging available information to select pediatric doses in rare disease. Additionally, challenges with translation and associated dose selection of new modalities such as gene therapy in rare diseases are examined with the lens of continuous learning and knowledge development that will enable pediatric dose selection of these modalities with confidence.

Evaluation of pharmacokinetics and safety of talazoparib in patients with advanced cancer and varying degrees of hepatic impairment.

AIM: This phase I study investigated talazoparib pharmacokinetics (PK) and safety in patients with advanced solid tumours and varying degrees of hepatic function. METHODS: Patients with advanced solid tumours and normal hepatic function or varying degrees of hepatic impairment (mild, moderate or severe, based on National Cancer Institute Organ Dysfunction Working Group classification) received talazoparib 0.5 mg once daily for 22 calendar days. Plasma and urine samples after single and multiple doses were collected and analysed for talazoparib using validated assays. Plasma PK data from all patients were analysed using the population PK method. Plasma and urine PK parameters in PK-evaluable patients were calculated using noncompartmental analysis (NCA). Safety was monitored in all enrolled patients. RESULTS: Thirty-eight patients were enrolled; 37 had ≥1 PK concentration, among which 17 were evaluable for NCA. Population PK analysis (n = 37) indicated no significant impact of hepatic function on apparent clearance (CL/F) of talazoparib. Baseline creatinine clearance was the only significant covariate on CL/F (α = 0.05). NCA of data (n = 17) showed no clear trend for increase in exposure on day 22 with worsening hepatic function. Talazoparib protein binding was comparable in patients with varying hepatic function. Talazoparib was generally well tolerated, and the safety profile observed in this study was consistent with the known safety profile of the drug. CONCLUSIONS: Hepatic impairment (mild, moderate or severe) has no impact on the PK of talazoparib. No dose modification is recommended for patients with advanced solid tumours and various degrees of hepatic impairment, and this labelling language has been approved by the US Food and Drug Administration and the European Medicines Agency.

Palbociclib has no clinically relevant effect on the QTc interval in patients with advanced breast cancer.

The aim of this study was to assess the potential effects of palbociclib in combination with letrozole on QTc. PALOMA-2, a phase 3, randomized, double-blind, placebo-controlled trial, compared palbociclib plus letrozole with placebo plus letrozole in postmenopausal women with estrogen receptor-positive, human epidermal growth factor receptor 2-negative advanced breast cancer. The study included a QTc evaluation substudy carried out as a definitive QT interval prolongation assessment for palbociclib. Time-matched triplicate ECGs were performed at 0, 2, 4, 6, and 8 h at baseline (Day 0) and on Cycle 1 Day 14. Additional ECGs were collected from all patients for safety monitoring. The QT interval was corrected for heart rate using Fridericia's correction (QTcF), Bazett's correction (QTcB), and a study-specific correction factor (QTcS). In total, 666 patients were randomized 2 : 1 to palbociclib plus letrozole or placebo plus letrozole. Of these, 125 patients were enrolled in the QTc evaluation substudy. No patients in the palbociclib plus letrozole arm of the substudy (N=77) had a maximum postbaseline QTcS or QTcF value of ≥ 480 ms, or a maximum increase from clock time-matched baseline for QTcS or QTcF values of ≥ 60 ms. The upper bounds of the one-sided 95% confidence interval for the mean change from time-matched baseline for QTcS, QTcF, and QTcB at all time points and at steady-state Cmax following repeated administration of 125 mg palbociclib were less than 10 ms. Palbociclib, when administered with letrozole at the recommended therapeutic dosing regimen, did not prolong the QT interval to a clinically relevant extent.

Ocular Pharmacokinetics.

Although the fundamental concepts of pharmacokinetics remain the same, ocular pharmacokinetics has its own challenges due to the uniqueness of barrier properties posed by various ocular tissues and its growing complexity with different routes of ocular administration. A thorough understanding of the barrier nature will aid in tailoring a drug or its carrier's physicochemical properties to its advantage. In order to deliver the right payload of a drug at the target site, various approaches can be taken to leverage the pharmacokinetics that includes molecular design based on desirable physicochemical properties, formulation approaches, and alternative routes of administration. In this chapter, a brief overview of the barrier properties with respect to various routes of administration is presented along with the physicochemical properties that influence the pharmacokinetics of ocular drugs. Recent advances in ocular pharmacokinetics are discussed in addition to new perspectives in interpreting existing data.

Mechanism - based translational pharmacokinetic - pharmacodynamic model to predict intraocular pressure lowering effect of drugs in patients with glaucoma or ocular hypertension.

PURPOSE: To develop a mechanism based translational pharmacokinetic-pharmacodynamic (PKPD) model in preclinical species and to predict the intraocular pressure (IOP) following drug treatment in patients with glaucoma or ocular hypertension (OHT). METHODS: Baseline diurnal IOP of normotensive albino rabbits, beagle dogs and patients with glaucoma or OHT was collected from literature. In addition, diurnal IOP of patients treated with brimonidine or Xalatan® were also obtained from literature. Healthy normotensive New Zealand rabbits were topically treated with a single drop of 0.15% brimonidine tartrate and normotensive beagle dogs were treated with a single drop of Xalatan®. At pre-determined time intervals, IOP was measured and aqueous humor samples were obtained from a satellite group of animals. Population based PKPD modeling was performed to describe the IOP data and the chosen model was extended to predict the IOP in patients. RESULTS: Baseline IOP clearly depicts a distinctive circadian rhythm in rabbits versus human. An aqueous humor dynamics based physiological model was developed to describe the baseline diurnal IOP across species. Model was extended to incorporate the effect of drug administration on baseline IOP in rabbits and dogs. The translational model with substituted human aqueous humor dynamic parameters predicted IOP in patients following drug treatment. CONCLUSIONS: A physiology based mechanistic PKPD model was developed to describe the baseline and post-treatment IOP in animals. The preclinical PKPD model was successfully translated to predict IOP in patients with glaucoma or OHT and can be applied in assisting dose and treatment selection and predicting outcome of glaucoma clinical trials.

International consortium for innovation and quality: An industry perspective on the nonclinical and early clinical development of intravitreal drugs.

The eye, which is under constant exposure to environmental pathogens, has evolved various anatomic and immunological barriers critical to the protection of tissues lacking regenerative capacity, and the maintenance of a clear optic pathway essential to vision. By bypassing the ocular barriers, intravitreal (IVT) injection has become the mainstay for the delivery of drugs to treat conditions that affect the back of the eye. Both small molecules and biotherapeutics have been successfully administered intravitreally, and several drugs have been approved for the treatment of (wet) age-related macular degeneration and diabetic macular edema. However, IVT injection is an invasive procedure, which requires sufficient technical expertise from the healthcare professional administering the drug. Potential side effects include bleeding, retinal tear, cataracts, infection, uveitis, loss of vision, and increased ocular pressure. Pharmaceutical companies often differ in their drug development plan, including drug administration techniques, collection of ocular tissues and fluids, ophthalmology monitoring, and overall conduct of nonclinical and clinical studies. The present effort, under the aegis of the Innovation & Quality Ophthalmic Working Group, aims at understanding these differences, identifying pros and cons of the various approaches, determining the gaps in knowledge, and suggesting feasible good practices for nonclinical and early clinical IVT drug development.

Intraocular distribution of melanin in human, monkey, rabbit, minipig and dog eyes.

The purpose of this study was to quantify the melanin pigment content in sclera, choroid-RPE, and retina, three tissues encountered during transscleral drug delivery to the vitreous, in human, rabbit, monkey, minipig, and dog models. Strain differences were assessed in NZW × NZR F1 and Dutch belted rabbits and Yucatan and Gottingen minipigs. The choroid-RPE and retina tissues were divided into central (posterior pole area) and peripheral (away from posterior pole) regions while the sclera was analyzed without such division. Melanin content in the tissues was analyzed using a colorimetric assay. In all species the rank order for pigment content was: choroid-RPE >retina ≥ sclera, except in humans, where scleral melanin levels were higher than retina and central choroid. The melanin content in a given tissue differed between species. Further, while the peripheral tissue pigment levels tended to be generally higher compared to the central regions, these differences were significant in human in the case of choroid-RPE and in human, monkey, and dogs in the case of retina. Strain difference was observed only in the central choroid-RPE region of rabbits (NZW × NZR F1 >Dutch Belted). Species, strain, and regional differences exist in the melanin pigment content in the tissues of the posterior segment of the eye, with Gottingen minipig being closest to humans among the animals assessed. These differences in melanin content might contribute to differences in drug binding, delivery, and toxicity.

Impact of Dose Reduction on Efficacy: Implications of Exposure-Response Analysis of Palbociclib.

BACKGROUND: Palbociclib is indicated for hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced breast cancer (ABC). OBJECTIVE: Exposure-response analyses were conducted to evaluate efficacy in Asian versus non-Asian patients and in patients with versus without dose reduction in PALOMA-2. PATIENTS AND METHODS: PALOMA-2 compared palbociclib plus letrozole versus placebo plus letrozole in patients with ABC. Population pharmacokinetic analysis provided apparent palbociclib clearance (CL/F) for each patient. The time-varying exposure metric, Cavg,t, was calculated using average dose intensity and CL/F at the time of each progression-free survival (PFS) event. A Cox proportional model characterized PFS and palbociclib Cavg,t relationships. Significant prognostic factors for PFS were identified by univariate analysis, which were subsequently included in multivariate analyses, in addition to the Cavg,t effect on PFS. PFS profiles in Asian/non-Asian patients and patients with/without dose reduction were simulated and compared using observed palbociclib exposures and established exposure-response relationships. RESULTS: Patients (n = 421) received palbociclib plus letrozole (Asian = 64, non-Asian = 357; no dose reduction = 272, dose reduction = 149). Based on univariate analyses, significant prognostic factors were Ki67 score, age, and baseline aspartate aminotransferase (BAST), tumor size, alkaline phosphatase, and albumin levels. In multivariate analysis, only Ki67 and BAST remained significant. Palbociclib exposure did not significantly affect PFS in either univariate (P = 0.12) or multivariate (P = 0.44) analyses. CONCLUSIONS: This analysis suggests that palbociclib exposure has no impact on PFS when the dose reduction algorithm from palbociclib clinical trials is used. There is no difference in efficacy between Asians and non-Asians, despite the higher level of dose reductions in Asians. PFIZER: NCT01740427.

The Effect of Renal Impairment on the Pharmacokinetics and Safety of Talazoparib in Patients with Advanced Solid Tumors.

BACKGROUND: Pharmacokinetic (PK) studies suggest that talazoparib is primarily eliminated unchanged via renal excretion. The current study investigated how varying degrees of renal impairment may affect the PK of talazoparib, and evaluated the safety and tolerability of talazoparib, in patients with advanced solid tumors with/without renal impairment. METHODS: Patients with advanced solid tumors and normal renal function or different degrees of renal impairment measured by estimated glomerular filtration rate (eGFR: mild = 60-89, moderate = 30-59, severe = 15-29 mL/min/1.73 m2) were enrolled in this open-label, non-randomized, phase I study. Talazoparib was administered orally at 0.5 mg/day for 22 days. Primary PK parameters included the area under the plasma concentration-time curve from 0 to 24 h (AUC0-24) and maximum observed plasma concentration (Cmax) at steady state (Day 22). Safety and tolerability were also investigated. RESULTS: Thirty-four patients were enrolled. At Day 22, compared with patients with normal renal function (n = 9), patients with mild (n = 9), moderate (n = 8), or severe (n = 8) renal impairment had a 12.2%, 43.0%, and 163.3% increase in talazoparib AUC0-24, and a 11.1%, 31.6%, and 89.3% increase in talazoparib Cmax, respectively. Talazoparib was generally well tolerated, and overall there were no notable differences in the treatment-emergent adverse event profile across renal function groups. CONCLUSIONS: Exposure to talazoparib increased with worsening renal impairment. Overall, this study confirms current dosing recommendations in patients with mild and moderate renal impairment (1 mg/day and 0.75 mg/day, respectively) and indicates that a lower starting dose of 0.5 mg/day should be considered for patients with severe renal impairment. CLINICAL TRIALS REGISTRATION: NCT02997163.

Population Pharmacokinetics of Talazoparib in Patients With Advanced Cancer.

Poly(ADP-ribose) polymerase (PARP) inhibitors have been developed to treat cancers associated with somatic BRCA mutations and germline genetic aberrations involved in the DNA damage response. The efficacy, tolerability, and pharmacokinetic/pharmacodynamic (PK/PD) profile of talazoparib, a potent small-molecule PARP inhibitor, was established in 4 clinical studies in cancer patients (2 phase 1 studies PRP-001 and PRP-002, the phase 2 ABRAZO trial, and the phase 3 EMBRACA trial). The current study aimed to describe the population PK of talazoparib and identify covariates that affect talazoparib PK in patients with advanced cancers using pooled data from these 4 studies. Talazoparib PK was well characterized by a 2-compartment model with first-order absorption and absorption lag time. Based on covariate analysis, no dose adjustment for talazoparib is required based on a patient's age, sex, baseline body weight, Asian race, the presence of mild renal or hepatic impairment, or use of acid-reducing agents. A reduced 0.75-mg daily dose is recommended for patients taking a potent P-glycoprotein inhibitor and those with moderate renal impairment. Insufficient data were available to establish dosing recommendations for patients with severe renal and moderate or severe hepatic impairment. The PK of a single 1-mg talazoparib capsule is comparable with 4 0.25-mg capsules. Talazoparib can be taken with or without food. These data provide support for dosing recommendations and labeling information for talazoparib.

Prediction of vitreal half-life based on drug physicochemical properties: quantitative structure-pharmacokinetic relationships (QSPKR).

PURPOSE: The aim of this study was to develop quantitative structure pharmacokinetic relationships (QSPKR) to correlate drug physicochemical properties (molecular weight, lipophilicity, and drug solubility), dose, salt form factor, and eye pigmentation factor to intravitreal half-life in the rabbit model. METHODS: Dataset derived from prior literature reports, which included molecules with complete structural diversity, was used to develop the QSPKR models. Entire dataset as well as subsets limited to albino rabbit data, pigmented rabbit data, acids, bases, zwitterions, neutral compounds, suspensions, and macromolecules were analyzed. Multiple linear regression analysis was carried out with noncollinear independent variables and the best-fit models were selected based on correlation coefficients and goodness of fit statistics. RESULTS: The analysis indicated that logarithm of MW (Log MW), lipophilicity (Log P or Log D) and dose number (dose/solubility at pH 7.4), are the most critical determinants of intravitreal half-life of the compounds analyzed. The best-fit models obtained from the entire dataset (Log t (1/2) = -0.178 + 0.267 Log MW - 0.093 Log D + 0.003 dose/solubility at pH 7.4 + 0.153 Pigmentation Factor and Log t (1/2) = -0.32 + 0.432 Log MW - 0.157 Log P + 0.003 dose/solubility at pH 7.4) predicted the various subsets well. Pigmented dataset and zwitterions were better predicted by Log P rather than Log D. CONCLUSIONS: The present study confirmed that intravitreal half-life could be better predicted by a group of variables (Log MW, Log P or Log D, dose number) rather than a single variable. In general, increasing Log MW and dose number, while reducing Log D or Log P would be beneficial for prolonging intravitreal half-life of drugs.

Human scleral diffusion of anticancer drugs from solution and nanoparticle formulation.

PURPOSE: To determine the transscleral permeability of chemotherapeutic drugs vinblastine and doxorubicin for treatment of intraocular tumors, and to compare the use of doxorubicin encapsulated in PLGA and liposome nanoparticles. METHODS: Human sclera was isolated and mounted in a Lucite chamber. Fluorescently tagged vinblastine (VIN), innately fluorescent free doxorubicin (DOX), PLGA doxorubicin (PLGA-DOX), or Doxil (Tibotec Therapeutics) were added to the episcleral donor chamber. The choroidal side was perfused with Balanced Salt Solution. Perfusate fractions were collected over 24 h and measured for fluorescence. Following the experiment, tissue sections were imaged, underwent a drug wash out procedure, and tissue drug content was analyzed using an LC-MS/MS method. RESULTS: Within 24 h, a total of 68%, 74%, 29%, and 1.9% of the drug dose from VIN, DOX, PLGA-DOX, and Doxil, respectively, diffused across the sclera. VIN and DOX scleral tissue showed strong fluorescence after 24 h. PLGA-DOX displayed scattered fluorescence, and Doxil indicated minimal fluorescence. LC-MS/MS revealed strong tissue binding of DOX. CONCLUSIONS: This study suggests both vinblastine and doxorubicin are able to diffuse across human sclera. In addition, PLGA nanoparticles delivered doxorubicin at a slower rate across the sclera, and the liposome preparation resulted in the slowest delivery of drug.

Distribution, Metabolism, and Excretion of Gedatolisib in Healthy Male Volunteers After a Single Intravenous Infusion.

In this open-label study (NCT02142920), we investigated the distribution, pharmacokinetics, and metabolism of the pan-class-I isoform phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor gedatolisib (PF-05212384), following a single intravenous administration in healthy male subjects. A single, 89-mg, intravenous dose of gedatolisib was associated with a favorable safety profile in the 6 healthy subjects evaluated. Peak plasma concentrations for unchanged gedatolisib and total radioactivity were observed at the end of the 30-minute infusion. The only observed drug-related material in plasma was the parent drug, gedatolisib. Terminal half-life for plasma gedatolisib was ∼37 hours. Following the dose, 66%-73% of drug-related material was recovered in the feces. Metabolism of gedatolisib was trace; only 1 oxidative metabolite, M5, was identified in feces (<1% of total dose). Identification of gedatolisib in feces suggests that biliary and/or intestinal secretion of unchanged parent drug significantly contributes to gedatolisib clearance.

Optimal sampling scheme for estimation of intraocular pressure diurnal curves in glaucoma trials.

BACKGROUND AND OBJECTIVE: Effective control of intraocular pressure (IOP) is essential for the successful management of glaucoma. IOP exhibits diurnal variation, yet continuous monitoring is impractical. To date, no clear evidence exists on the number of sampling timepoints required to characterize diurnal IOP and when those measurements should be collected. The objective of this study was to develop an optimized sampling scheme to estimate diurnal IOP and to provide sampling windows for practicality. METHODS: Baseline IOP values for glaucoma patients were collected from the published literature. A population model-based meta-analysis was performed to develop a model for diurnal IOP that accounts for covariates and inter-study variability. Optimization was performed using the D-optimality criteria to determine optimal sampling times. In addition, various reduced sampling designs were tested to investigate the minimum number of sampling timepoints to precisely estimate diurnal IOP. Also, sampling windows were calculated around the final optimal sampling times to allow flexibility in data collection. The final reduced optimized model was validated by simulating and estimating 500 datasets with reduced optimal sampling times. RESULTS: The final baseline IOP model included type of glaucoma as a covariate. Bootstrap analysis and visual predictive check plots revealed the adequacy of the model to describe the observed IOP data. Optimization results indicated an increasing trend in bias with decreasing sampling timepoints. A reduced model with four sampling times resulted in acceptable precision (<40 %). Restricting the sampling time between 8 a.m. and 4 p.m. underestimates the fluctuation in diurnal IOP. Sampling windows with ≥95 % efficiency were calculated around the optimized sampling times. Validation results indicated acceptable precision and relative bias for model estimates in the reduced optimized model. CONCLUSION: A physiologically based mechanistic model was developed to describe the diurnal variation in baseline IOP and inter-study variability was estimated on key diurnal model parameters. Optimization of the final covariate model indicated a reduced sampling time of at least four samples should be collected at 5:45 a.m., 2:15 p.m., 8:00 p.m., and 12:00 a.m. for reliable estimation of diurnal IOP variation.

Comparison of long-acting bevacizumab formulations in the treatment of choroidal neovascularization in a rat model.

OBJECTIVE: The objective of this study was to compare the reduction in size of experimentally induced choroidal neovascularization (CNV) in rat eyes treated with bevacizumab, poly(ethylene-glycol) (PEG)-bevacizumab conjugate (b-PEG), and poly(lactic-co-glycolic acid) (PLGA)-encapsulated bevacizumab (b-PLGA). METHODS: Forty-eight eyes from 24 rats were divided into 4 groups of 12 eyes. In each group, 3 eyes were assigned to a treatment subgroup, each receiving a different injection-control, bevacizumab, b-PEG, and b-PLGA. In all eyes, laser photocoagulation was used to rupture Bruch's membrane. In group 1, laser was followed by injection, which was then followed by harvesting the rats to assess the CNV area. All 3 steps were separated by a 2-week interval. In groups 2, 3, and 4, injection preceded laser photocoagulation by a variable interval and all rats were harvested 2 weeks postlaser treatment. In group 2, laser and injection were separated by 2 weeks. In group 3, laser followed injection by 4 weeks. In group 4, laser followed injection by 6 weeks. The CNV area was measured for each subgroup and compared against its control. Pairwise comparisons were conducted to assess for statistically significant differences between subgroups. RESULTS: All subgroups in groups 1, 2, and 4 showed statistically significant reduction of CNV area (P<0.05). In group 3, the b-PEG and b-PLGA subgroups showed a 9.0% (P=0.384) and 20.3% (P=0.077) reduction in CNV area versus control, whereas there was no reduction in CNV area in the bevacizumab subgroup. However, this was not found to be statistically significant. In group 4, b-PEG was more effective than bevacizumab and b-PLGA. CONCLUSION: The reduction in CNV area in all treatment subgroups, with the exception of those in group 3, suggests successful creation of the 2 bevacizumab formulations while retaining its active antiangiogenic properties. Further studies varying in dosages and timing of injection and laser are needed to evaluate the formulations' long-acting efficacy.

Nanosized dendritic polyguanidilyated translocators for enhanced solubility, permeability, and delivery of gatifloxacin.

PURPOSE: Dendrimeric polyguanidilyated translocators (DPTs) are nanosized novel dendrimers that efficiently translocate molecules across biological barriers. The purpose of this study was to develop a DPT that could serve as an ophthalmic delivery vehicle for gatifloxacin and to evaluate its in vitro and in vivo delivery after topical application. METHODS: The gatifloxacin (GFX) solubility-enhancing property of a six-guanidine group-containing dendrimer (g6 DPT) was investigated as a function of pH and dendrimer concentration. Mechanisms of drug interaction with the dendrimer were investigated by using isothermal titration calorimetry (ITC), Fourier-transformed infrared spectroscopy (FTIR), and nuclear magnetic resonance spectroscopy (NMR). Permeability of the dendrimer was assessed in human corneal epithelial cells (HCECs) and across isolated bovine sclera-choroid-RPE (SCRPE). In vitro efficacy of the dendrimer formulation was evaluated with a time-to-kill assay for methicillin resistant Staphylococcus aureus (MRSA). In vivo delivery of GFX in a dendrimer eye drop formulation was studied in New Zealand White rabbits after a single dose or multiple doses over 3 weeks. Drug levels in various ocular tissues were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: An optimized DPT-GFX formulation (final pH 5.9, no preservative) increased GFX solubility by fourfold. The dendrimer formed isotonically stable, nanosized (346-nm) complexes with GFX via ionic bond, hydrogen bond, and hydrophobic interactions. The dendrimer gained rapid entry into the HCECs (within 5 minutes) and increased the transport of GFX by 40% across the SCRPE in 6 hours. DPT-GFX exhibited a three times faster killing rate for MRSA when compared with GFX alone. In vivo administration of DPT-GFX (1.2% wt/vol) resulted in ∼13-fold, and ∼2-fold higher areas under the curve (AUCs) for tissue concentrations in conjunctiva and cornea, respectively, when compared with GFX (0.3%) after a single dose. Further, a single dose of DPT-GFX sustained aqueous humor and vitreous humor drug levels during the 24-hour study, with a t(1/2) of 9 and 32 hours, respectively. After multiple doses, similar advantages were seen with DPT-GFX. CONCLUSIONS: The DPT forms stable complexes with GFX and enhances its solubility, permeability, anti-MRSA activity, and in vivo delivery, potentially allowing a once-daily dose regimen.

Luteinizing hormone-releasing hormone receptor-targeted deslorelin-docetaxel conjugate enhances efficacy of docetaxel in prostate cancer therapy.

Docetaxel, a chemotherapeutic agent currently used for improving survival of prostate cancer patients, suffers from low therapeutic index. The objective of this study was to prepare a new docetaxel derivative conjugated to deslorelin, a luteinizing hormone-releasing hormone (LHRH) superagonist, and to determine whether it enhances docetaxel potency in vitro and in vivo. Because docetaxel is not amenable for conjugation with peptides, we introduced a -COOH group in docetaxel, forming docetaxel-hemiglutarate, and subsequently conjugated this to serine in deslorelin, forming deslorelin-docetaxel. Fourier-transform IR, (1)H-nuclear magnetic resonance, and liquid chromatography-mass spectrometry analyses confirmed deslorelin-docetaxel formation. Antiproliferative efficacy in LNCaP and PC-3 cell lines over 24, 48, and 72 hours exhibited the order deslorelin-docetaxel > docetaxel, whereas deslorelin alone had no effect, with deslorelin-docetaxel potency being 15-fold greater than docetaxel at 72 h. Further, cells pretreated with antisense oligonucleotide against LHRH receptor exhibited decreased deslorelin-docetaxel efficacy, without any change in docetaxel efficacy. Thus, deslorelin-docetaxel efficacy is likely mediated via LHRH receptor. Cell cycle analysis showed that docetaxel treatment led to arrest in G(2)-M phase, whereas deslorelin-docetaxel treatment allowed greater progression to apoptosis in both cell lines, with deslorelin-docetaxel exerting 5-fold greater apoptosis compared with docetaxel in prostate cancer cell lines. Antitumor efficacy studies in PC-3 prostate xenograft-bearing mice indicated the efficacy order deslorelin-docetaxel > docetaxel >> deslorelin > PBS, with deslorelin-docetaxel exerting approximately 5.5-fold greater tumor growth inhibition than docetaxel alone. Thus, deslorelin-docetaxel prepared in this study retains pharmacologic effects of both docetaxel and deslorelin while enhancing the antiproliferative, apoptotic, and antitumor efficacy of docetaxel by several folds in prostate cancer therapy.

Subconjunctival nanoparticle carboplatin in the treatment of murine retinoblastoma.

OBJECTIVE: To evaluate the efficacy of subconjunctival nanoparticle carboplatin in the treatment of transgenic murine retinoblastoma. METHODS: Dendrimeric nanoparticles loaded with carboplatin were prepared. Forty LHbeta-Tag mice were randomly assigned into 4 groups and treated at 10 weeks of age. Each mouse received a single subconjunctival injection in one eye, and the opposite eye was left untreated as a control. Group 1 (high-dose nanoparticle carboplatin) received 37.5 mg/mL of nanoparticle carboplatin; group 2 (low-dose nanoparticle carboplatin) received 10 mg/mL of nanoparticle carboplatin; group 3 (conventional carboplatin) received 10 mg/mL of carboplatin in aqueous solution; and group 4 (phosphate-buffered saline) received phosphate-buffered saline. Mice were killed on day 22 after treatment. Eyes were serially sectioned, and retinal tumor burden was quantified by histopathologic analysis. RESULTS: Mean tumor burden in the treated eyes was significantly smaller compared with the untreated eyes in the same mice in both nanoparticle carboplatin groups (group 1, P = .02; group 2, P = .02) and the treated eyes in the conventional carboplatin group (group 1 vs group 3, P < .01; group 2 vs group 3, P = .01) and phosphate-buffered saline group (group 1 vs group 4, P < .01; group 2 vs group 4, P = .01). The untreated eyes in the high-dose nanoparticle carboplatin group showed significantly smaller tumor mass compared with the conventional carboplatin (P = .03) and PBS (P = .04) groups. No toxic effects were observed in any of the groups. CONCLUSION: A single injection of subconjunctival nanoparticle carboplatin was effective in the treatment of transgenic murine retinoblastoma, with no associated toxic effects. The higher dose of subconjunctival nanoparticle carboplatin decreased the tumor burden in the contralateral eye. CLINICAL RELEVANCE: This model provides a basis to test carboplatin nanoparticles for the treatment of human retinoblastoma.

Influence of dosage form on the intravitreal pharmacokinetics of diclofenac.

PURPOSE: To prepare a suspension form of diclofenac and compare the influence of the injected form (suspension versus solution) on the intravitreal pharmacokinetics of diclofenac in Dutch belted pigmented rabbits. METHODS: Diclofenac acid was prepared and characterized in a suspension formulation. Rabbit eyes were injected with either diclofenac sodium solution (0.3 mg) or diclofenac acid suspension (10 mg) prepared in 0.1 mL balanced salt solution. Rabbits were killed at regular time intervals, the eyes enucleated, and drug content quantified in the vitreous humor and retina-choroid tissue by high-performance liquid chromatography. Pharmacokinetic models were developed for both the dosage forms, and simulations were performed for different doses. RESULTS: Diclofenac acid with an approximate 5-mum particle size exhibited 3.5-fold lower solubility in vitreous humor, when compared with its sodium salt. The estimated settling velocity of the suspension in the vitreous humor was 3 cm/h. After diclofenac sodium salt solution injection, drug levels declined rapidly with no drug levels detectable after 24 hours in the vitreous humor and 4 hours in the RC. Throughout the assessed time course, drug levels were higher in the vitreous. However, sustained, high drug levels were observed in both the vitreous humor and the retina-choroid even on day 21 after diclofenac acid suspension injection, with retina-choroid drug levels being higher beginning at 0.25 hour. The elimination half-life of diclofenac suspension was 24 and 18 days in vitreous and retina-choroid, respectively, compared to 2.9 and 0.9 hours observed with diclofenac sodium. The pharmacokinetic models developed indicated a slow-release distribution or depot compartment for the diclofenac acid suspension in the posterior segment. Simulations indicated the inability of a 10-mg dose of diclofenac sodium solution to sustain drug levels in the vitreous beyond 11 days. CONCLUSIONS: By choosing a less soluble form of a drug such as diclofenac acid, vitreous elimination half-life can be prolonged up to 24 days, potentially resulting in therapeutic levels in the posterior segment tissues for a few months. Higher detectable drug levels in the retina-choroid suggest rapid settling and persistent retention of suspension in retina-choroid tissue.

Targeted drug and gene delivery systems for lung cancer therapy.

PURPOSE: To evaluate the efficacy of a novel docetaxel derivative of deslorelin, a luteinizing hormone-releasing hormone (LHRH) agonist, and its combination in vivo with RGD peptide conjugated nanoparticles encapsulating an antiangiogenic, anti-vascular endothelial growth factor (VEGF) intraceptor (Flt23k; RGD-Flt23k-NP) in H1299 lung cancer cells and/or xenografts in athymic nude BALB/c mice. EXPERIMENTAL DESIGN: The in vitro and in vivo efficacy of the deslorelin-docetaxel conjugate was evaluated in H1299 cells and xenografts in athymic nude mice. Coadministration of deslorelin-docetaxel conjugate and RGD-Flt23k-NP was tested in vivo in mice. Tumor inhibition, apoptosis, and VEGF inhibition were estimated in each of the treatment groups. RESULTS: The conjugate enhanced in vitro docetaxel efficacy by 13-fold in H1299 cells compared with docetaxel at 24 hours, and this effect was inhibited following reduction of LHRH receptor expression by an antisense oligonucleotide. Combination of the conjugate with the RGD-Flt23k-NP in vivo resulted in an 82- and 15-fold tumor growth inhibition on day 39 following repeated weekly i.v. injections and a single intratumoral (i.t.) injection, respectively. These effects were significantly greater than individual targeted therapies or docetaxel alone. Similarly, apoptotic indices for the combination therapy were 14% and 10% in the i.v. and i.t. groups, respectively, and higher than the individual therapies. Combination therapy groups exhibited greater VEGF inhibition in both the i.v. and i.t. groups. CONCLUSIONS: Docetaxel efficacy was enhanced by LHRH receptor-targeted deslorelin conjugate and further improved by combination with targeted antiangiogenic nanoparticle gene therapy. Combination of novel targeted therapeutic approaches described here provides an attractive alternative to the current treatment options for lung cancer therapy.