Preclinical Studies of Gene Replacement Therapy for CDKL5 Deficiency Disorder.

Cyclin dependent kinase-like 5 (CDKL5) Deficiency Disorder (CDD) is a rare neurodevelopmental disorder caused by a mutation in the X-linked CDKL5 gene. CDKL5 is a serine/threonine kinase that is critical for axon outgrowth, dendritic morphogenesis, as well as synapse formation, maturation, and maintenance. This disorder is characterized by early-onset epilepsy, hypotonia, and failure to reach cognitive and motor developmental milestones. Because the disease is monogenic, delivery of the CDKL5 gene to the brain of patients should provide clinical benefit. To this end, we designed a gene therapy vector, adeno-associated virus (AAV)9.Syn.hCDKL5, in which human CDKL5 gene expression is driven by the synapsin promoter. In biodistribution studies conducted in mice, intracerebroventricular (ICV) injection resulted in broader, more optimal biodistribution than did intracisterna magna (ICM) delivery. AAV9.Syn.hCDKL5 treatment increased phosphorylation of EB2, a bona fide CDKL5 substrate, demonstrating biological activity in vivo. Our data provides proof-of-concept that ICV delivery of AAV9.Syn.hCDKL5 to neonatal male Cdkl5 knockout mice reduces pathology and reduces aberrant behavior. Functional improvements were seen at doses of 3e11 to 5e11 vector genomes (vg)/g brain, which resulted in transfection of ≥50% of the neurons. Functional improvements were not seen at lower doses suggesting a requirement for broad distribution for efficacy.

Comparison of pharmaceutical properties and biological activities of prednisolone, deflazacort, and vamorolone in DMD disease models.

Duchenne muscular dystrophy (DMD) is a progressive disabling X-linked recessive disorder that causes gradual and irreversible loss of muscle, resulting in early death. The corticosteroids prednisone/prednisolone and deflazacort are used to treat DMD as the standard of care; however, only deflazacort is FDA approved for DMD. The novel atypical corticosteroid vamorolone is being investigated for treatment of DMD. We compared the pharmaceutical properties as well as the efficacy and safety of the three corticosteroids across multiple doses in the B10-mdx DMD mouse model. Pharmacokinetic studies in the mouse and evaluation of p-glycoprotein (P-gP) efflux in a cellular system demonstrated that vamorolone is not a strong P-gp substrate resulting in measurable central nervous system (CNS) exposure in the mouse. In contrast, deflazacort and prednisolone are strong P-gp substrates. All three corticosteroids showed efficacy, but also side effects at efficacious doses. After dosing mdx mice for two weeks, all three corticosteroids induced changes in gene expression in the liver and the muscle, but prednisolone and vamorolone induced more changes in the brain than did deflazacort. Both prednisolone and vamorolone induced depression-like behavior. All three corticosteroids reduced endogenous corticosterone levels, increased glucose levels, and reduced osteocalcin levels. Using micro-computed tomography, femur bone density was decreased, reaching significance with prednisolone. The results of these studies indicate that efficacious doses of vamorolone, are associated with similar side effects as seen with other corticosteroids. Further, because vamorolone is not a strong P-gp substrate, vamorolone distributes into the CNS increasing the potential CNS side-effects.

CHL1-Deficient and Wild-Type Male Mice do Not Differ in Locomotor Recovery from Spinal Cord Injury.

CHL1 is a close homolog of L1, a cell adhesion molecule that plays major roles in neural and tumor cell functions. We had found that young adult female mice deficient in CHL1 recovered better than their wild-type female littermates after thoracic Spinal Cord Injury (SCI). This observation was surprising, because CHL1 increases neurite outgrowth in vitro. Injury of adult mouse central and peripheral nervous systems upregulate CHL1 expression in neurons and astrocytes, consistent with CHL1's pro-active, homophilic interaction between CHL1 surface molecules in wild-type mice. After SCI, CHL1 expression was observed to increase in the glial scar, areas of axonal regrowth and remodeling of neural circuits. These observations were made only in females, and we therefore sought to analyze SCI in CHL1-deficient male mice. We now show that CHL1-deficient males did not recover better or worse than their male wild-type littermates. Primary and secondary lesion volumes were similar in the two genotypes, as seen in female mice which were studied in parallel with male mice. Assessment of peripheral leukocytes showed a significant increase in numbers of blood neutrophils at 24 h after SCI in males, but not in females. Lymphocyte numbers in mutant males increased slightly, but numbers of lymphocytes or monocytes did not differ significantly between males or females. These results indicate that CHL1-deficient males and females differ in the number of neutrophils but not lymphocytes or monocytes, suggesting that the difference between males and females is unlikely due to differences in leukocytes.

A sciatic nerve gap-injury model in the rabbit.

There has been an increased number of studies of nerve transection injuries with the sciatic nerve gap-injury model in the rabbit in the past 2 years. We wanted to define in greater detail what is needed to test artificial nerve guides in a sciatic nerve gap-injury model in the rabbit. We hope that this will help investigators to fully exploit the robust translational potential of the rabbit sciatic nerve gap-injury model in its capacity to test devices whose diameter and length are in the range of those commonly applied in hand and wrist surgery (diameter ranging between 2 and 4 mm; length up to 30 mm). We suggest that the rabbit model should replace the less translational rat model in nerve regeneration research. The rabbit sciatic model, however, requires an effective strategy to prevent and control self-mutilation of the foot in the postoperative period, and to prevent pressure ulcers.

Administration in fed state but not controlled release in the colon increases oral bioavailability of DF030263, a promising drug candidate for chronic lymphocytic leukemia.

For treatment of chronic cancers, the oral administration route is preferred as it provides numerous advantages over other delivery routes. However, these benefits of oral chemotherapy can be limited due to unfavorable pharmacokinetics. Accordingly, pharmacokinetic development of chemotherapeutic agents is crucial to the improvement of cancer treatment. In this study, assessment and optimization of biopharmaceutical properties of a promising drug candidate for cyclin-dependent kinase 9 (CDK9) inhibitor (DF030263) was performed to promote oral delivery. Oral bioavailability of DF030263 in fasted rats was 23.8%, and a distinct double-peak phenomenon was observed. A two-site absorption windows mechanism was proposed as a possible explanation to the phenomenon. The two-site absorption window hypothesis was supported by in vitro solubility assays in biorelevant fluids with different pH levels, as well as by in silico simulation by GastroPlus™. Controlled release to the colon was conducted in rats in order to exploit the colonic absorption window but did not improve the oral bioavailability. On the other hand, oral administration at postprandial conditions in rats (performed based on the high in vitro solubility in fed state simulated fluid and reduced pH-dependency) resulted in an almost 3-fold increase in bioavailability to 63.6%. In conclusion, this study demonstrates an efficient in vitro-in vivo-in silico drug development approach for improving the oral bioavailability of DF030263, a promising candidate for the treatment of chronic lymphocytic leukemia.

Lymphatic Distribution of Etanercept Following Intravenous and Subcutaneous Delivery to Rats.

PURPOSE: The purpose of this work was to investigate the role of the lymphatic system in the pharmacokinetics of etanercept, a fusion protein. METHODS: Etanercept 1 mg/kg was administered intravenously (IV) and subcutaneously (SC) to thoracic lymph duct-cannulated and sham-operated control rats. Blood and lymph samples were obtained for up to 6 days. RESULTS: Model-based SC bioavailability of etanercept was 65.2% in the control group. In lymph-cannulated rats, etanercept concentration in the lymph was consistently lower than in serum following IV dosing; and the concentration in the lymph was significantly higher than in serum after SC injection. The absorption occurred predominantly through the lymphatic pathway (82.7%), and only 17.3% by direct uptake into the central compartment (blood pathway). Lymphatic cannulation reduced the area under the serum concentration-time curve by 28% in IV group and by 91% in SC group. A mechanistic pharmacokinetic model that combined dual absorption pathways with redistribution of the systemically available protein drug into lymph was developed. The model successfully captured serum and lymph data in all groups simultaneously, and all parameters were estimated with sufficient precision. CONCLUSIONS: Lymphatic system was shown to play an essential role in systemic disposition and SC absorption of etanercept.

Is rat a good model for assessment of particulate-based taste-masked formulations?

Recently there has been an increased interest to develop specialised dosage forms that are better suited to specific patient populations, such as paediatrics and geriatrics. In these patient populations the acceptability of the oral dosage form can be paramount to the products success. However, many Active Pharmaceutical Ingredients (APIs) are known to cause an aversive taste response. One way to increase the acceptability and to enhance the palatability of the formulation is to design coated taste-masked particulate-based dosage forms. The masking of poorly tasting drugs with physical barriers such as polymer coatings can be utilised to prevent the release of drug within the oral cavity, thus preventing a taste response. However, currently, there are few assessment tools and models available to test the efficiency of these particulate-based taste-masked formulations. The rat brief access taste aversion model has been shown to be useful in assessment of taste for liquid dosage forms. However, the applicability of the rat model for particulate-based taste masked formulations is yet to be assessed. It is not understood whether dissolution, solubility and thus exposure of the drug to taste receptors would be the same in rat and human. Therefore, rat saliva must be compared to human saliva to determine the likelihood that drug release would be similar within the oral cavity for both species. In this study rat saliva was characterised for parameters known to be important for drug dissolution, such as pH, buffer capacity, surface tension, and viscosity. Subsequently dissolution of model bitter tasting compounds, sildenafil citrate and efavirenz, in rat saliva was compared to dissolution in human saliva. For all parameters characterised and for the dissolution of both drugs in rat saliva, a substantial difference was observed when compared to human saliva. This discrepancy in saliva parameters and dissolution of model drugs suggests that preclinical taste evaluation of particulate-based taste-masked formulations suggests rat is not a good model for predicting taste of solid dosage forms or undissolved drug where dissolution is required. Alternative preclinical in vivo models in other species, or improved biorelevant in vitro models should be considered instead.

A method to deliver patterned electrical impulses to Schwann cells cultured on an artificial axon.

Information from the brain travels back and forth along peripheral nerves in the form of electrical impulses generated by neurons and these impulses have repetitive patterns. Schwann cells in peripheral nerves receive molecular signals from axons to coordinate the process of myelination. There is evidence, however, that non-molecular signals play an important role in myelination in the form of patterned electrical impulses generated by neuronal activity. The role of patterned electrical impulses has been investigated in the literature using co-cultures of neurons and myelinating cells. The co-culturing method, however, prevents the uncoupling of the direct effect of patterned electrical impulses on myelinating cells from the indirect effect mediated by neurons. To uncouple these effects and focus on the direct response of Schwann cells, we developed an in vitro model where an electroconductive carbon fiber acts as an artificial axon. The fiber provides only the biophysical characteristics of an axon but does not contribute any molecular signaling. In our "suspended wire model", the carbon fiber is suspended in a liquid media supported by a 3D printed scaffold. Patterned electrical impulses are generated by an Arduino 101 microcontroller. In this study, we describe the technology needed to set-up and eventually replicate this model. We also report on our initial in vitro tests where we were able to document the adherence and ensheath of human Schwann cells to the carbon fiber in the presence of patterned electrical impulses (hSCs were purchased from ScienCell Research Laboratories, Carlsbad, CA, USA; ScienCell fulfills the ethic requirements, including donor's consent). This technology will likely make feasible to investigate the response of Schwann cells to patterned electrical impulses in the future.

Lipophilic activated ester prodrug approach for drug delivery to the intestinal lymphatic system.

The intestinal lymphatic system plays an important role in the pathophysiology of multiple diseases including lymphomas, cancer metastasis, autoimmune diseases, and human immunodeficiency virus (HIV) infection. It is thus an important compartment for delivery of drugs in order to treat diseases associated with the lymphatic system. Lipophilic prodrug approaches have been used in the past to take advantage of the intestinal lymphatic transport processes to deliver drugs to the intestinal lymphatics. Most of the approaches previously adopted were based on very bulky prodrug moieties such as those mimicking triglycerides (TG). We now report a study in which a lipophilic prodrug approach was used to efficiently deliver bexarotene (BEX) and retinoic acid (RA) to the intestinal lymphatic system using activated ester prodrugs. A range of carboxylic ester prodrugs of BEX were designed and synthesised and all of the esters showed improved association with chylomicrons, which indicated an improved potential for delivery to the intestinal lymphatic system. The conversion rate of the prodrugs to BEX was the main determinant in delivery of BEX to the intestinal lymphatics, and activated ester prodrugs were prepared to enhance the conversion rate. As a result, an 4-(hydroxymethyl)-1,3-dioxol-2-one ester prodrug of BEX was able to increase the exposure of the mesenteric lymph nodes (MLNs) to BEX 17-fold compared to when BEX itself was administered. The activated ester prodrug approach was also applied to another drug, RA, where the exposure of the MLNs was increased 2.4-fold through the application of a similar cyclic activated prodrug. Synergism between BEX and RA was also demonstrated in vitro by cell growth inhibition assays using lymphoma cell lines. In conclusion, the activated ester prodrug approach results in efficient delivery of drugs to the intestinal lymphatic system, which could benefit patients affected by a large number of pathological conditions.

Plasma lipid profiling across species for the identification of optimal animal models of human dyslipidemia.

In an attempt to understand the applicability of various animal models to dyslipidemia in humans and to identify improved preclinical models for target discovery and validation for dyslipidemia, we measured comprehensive plasma lipid profiles in 24 models. These included five mouse strains, six other nonprimate species, and four nonhuman primate (NHP) species, and both healthy animals and animals with metabolic disorders. Dyslipidemic humans were assessed by the same measures. Plasma lipoprotein profiles, eight major plasma lipid fractions, and FA compositions within these lipid fractions were compared both qualitatively and quantitatively across the species. Given the importance of statins in decreasing plasma low-density lipoprotein cholesterol for treatment of dyslipidemia in humans, the responses of these measures to simvastatin treatment were also assessed for each species and compared with dyslipidemic humans. NHPs, followed by dog, were the models that demonstrated closest overall match to dyslipidemic humans. For the subset of the dyslipidemic population with high plasma triglyceride levels, the data also pointed to hamster and db/db mouse as representative models for practical use in target validation. Most traditional models, including rabbit, Zucker diabetic fatty rat, and the majority of mouse models, did not demonstrate overall similarity to dyslipidemic humans in this study.

Comparative analysis of the effects of antimuscarinic agents on bladder functions in both nonhuman primates and rodents.

Both the physiological role of muscarinic receptors for bladder function and the therapeutic efficacy of antimuscarinic agents for overactive bladder syndrome are well documented. We investigated the effect of antimuscarinic agents with different subtype selectivity on urodynamic parameters in nonhuman primates and rodents and compared plasma levels of these agents between species. Anesthetized rhesus monkeys were transurethrally catheterized, and the bladder was infused with saline. Urodynamic parameters were measured before and after intravenous drug administration. Tolterodine (nonselective) and oxybutynin (moderately M(3)-selective) increased bladder capacity at lower doses than those required to decrease micturition pressure. However, higher doses of darifenacin (M(3)-selective) were needed to increase the bladder capacity than those needed to decrease the micturition pressure. In rats, tolterodine had no effect on the bladder capacity but decreased the micturition pressure at all of the doses administered. Oxybutynin also decreased micturition pressure and increased bladder capacity at the highest dose. Plasma levels of these drugs overlap in both species. These results suggest that, in addition to the M(3) receptor, other muscarinic receptor subtypes contribute to regulate bladder storage function in nonhuman primates, since less subtype-selective tolterodine and oxybutynin showed higher specificity to the bladder capacity effect than the effect on micturition pressure compared with M(3)-selective darifenacin. In addition, the role of muscarinic receptors in bladder storage function varies between primates and rodents. Compared with rodents, muscarinic receptors may play a more active role during the storage phase to regulate the functional bladder capacity in primates.

Use of a rigid endoscope to teach urethral catheterization of the female dog.

Urethral catheterization of the female dog is known to be a challenging procedure. The authors describe a catheterization technique in which they use a rigid endoscope to visualize the canine urogenital vestibule during the procedure. The technique is particularly helpful as a training tool for students who are not yet experienced with the canine anatomy. The endoscope is attached to a video camera and monitor, allowing others to observe and learn the procedure.