DMPK/ADME
We are experienced at conducting in vivo pharmacokinetic and metabolism studies. Our in vivo proof of concept testing and model development is designed to confirm drug delivery, efficacy, and tolerance in rodents and rabbits.
What we can do for you:
- pharmacokinetic analysis of blood (plasma)
- metabolite isolation and characterization
- bioequivalence studies
- formulation screening and optimization
- bioavailability studies (po, sc, im, ip, intratracheal)
- single dose and multiple dose PK studies
- discovery liquid formulation development and optimization
- drug distribution in tissue/organ and body fluid and determination of blood and brain ratio for brain penetration
- metabolic kinetics with active metabolites
- PK study for design of dose and dose regimen in various rodent disease models
Ocular PD/Efficacy
Our ophthalmic services begin, run and conclude with a high level of technical expertise.
From specialized ocular dosing by trained scientists, to the precise dissection of specific ocular tissues by our specialists, followed by exacting sample processing and bioanalysis by PharmOptima’s experienced staff; we stand out against the large CRO model of generalized necropsy technicians and crash & shoot discovery bioanalysis. Our bioanalytical team has extensive experience processing, homogenizing and extracting ocular fluids and tissues. We have developed LC-MS/MS methods for hundreds of compounds.
What we can do for you:
- validate bioanalytical methods following current FDA Crystal City and OECD guidances
- perform cross validations for additional species and matrices
- assay plasma and dosing solutions from regulated animal safety studies (i.e. GLP) and clinical trials
- assay metabolism of drugs/prodrugs in ocular tissue
Optic Nerve Crush
Central nervous system trauma and neurodegenerative disorders can trigger a cascade of cellular and molecular events culminating in neuronal apoptosis. The Optic Nerve Crush model provides an effective tool for analyzing the pathogenic mechanisms associated with neuronal injury signaling in vivo. Optic nerve crush has been used as a model neuronal injury, including glaucoma, traumatic optic neuropathies, neurodegeneration and CNS injury. Crush injury to the optic nerve severs the retinal ganglion cell (RGC) axons leading to the gradual death of RGC neurons in the retina. The model provides an opportunity to study neuronal outcomes following injury, including survival, apoptosis, regeneration and associated biomarkers. Applications include traumatic optic neuropathy, glaucoma and neurodegenerative disease.
Optic nerve crush serves as a useful model for traumatic optic neuropathy and mimics glaucomatous injury, similarly inducing RGC cell death and degeneration. Glaucomatous injury is a pathohistological feature of glaucoma in the optic nerve.
Molecular Readouts Illustration Model Induction
Multidimensional observations strengthen the interpretation: in addition to in-life measurements (i.e., ERG), Immunostaining monitors therapeutic effect, immunoassays track biomarkers, and qRT-PCR provides information on retinal gene expression. Markers tracked in this model include:
| Protein/Gene | Significance |
|---|---|
| pcJun | neuronal injury |
| TUJI | RGC marker |
| Atf3 | regeneration-associated genes |
| Sprr1a | regeneration-associated genes |
| Ddit3 (Chop) | pro-apoptotic transcription factor |
| Gfap | Reactive astrocyte marker |
Treating and Reversing Glaucoma
The GD3 Ocular Center of Excellence is proud to offer efficacy models in which physiological readouts coupled with cellular and biochemical measurements provide a comprehensive snapshot of your treatment's therapeutic potential. The optic nerve crush model can test agents treating glaucoma, traumatic optic neuropathies, neurodegeneration, and CNS injury and inflammation. If your organization is working to treat any of these debilitating diseases, we encourage you to examine our capabilities:
- Glaucoma
- In vivo Mouse Model for Glaucoma
- Traumatic Optic Neuropathies
- CNS Injury
- Inflammation
- Neurodegenerative Diseases
Activation of Signaling Pathways
Western blot of retinal tissue three days following optic nerve crush compared to uninjured control: upregulation of injury marker, pcJun, demonstrates activation of signaling pathways important for neuronal outcome following ONC.
Upregulation of Injury Markers
Immunostained whole mount retinas following optic nerve crush (ONC): upregulation of injury marker, pcJun, demonstrates activation of injury signaling pathways resulting in retinal ganglion cell (RGC) death following ONC. Loss of pcJun and TUJI signal three weeks after ONC demonstrates a reduction in the number of surviving RGCs in the weeks following axotomy.
Robust Transcriptional Response
qRT-PCR of Atf3, Sprr1a, Ddit3 (Chop), and Gfap from retinal RNA four days after optic nerve crush (ONC) compared to uninjured contralateral control (CTL): upregulation of regeneration-associated genes Atf3 and Sprr1a, pro-apoptotic transcription factor Ddit3 (Chop), and reactive astrocyte marker Gfap demonstrates a robust response to injury following ONC. Relative gene expression was calculated using the ΔΔCT method relative to Gapdh and normalized to expression levels in CTL samples.
References
- Dual leucine zipper kinase-dependent PERK activation contributes to neuronal degeneration following insult. Larhammar et al. eLife 2017; 6:e20725.
- Longitudinal Morphological and Functional Assessment of RGC Neurodegeneration After Optic Nerve Crush in Mouse. Li et al. Front. Cell. Neurosci. 2020; 14 (109).
- An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival. Tang et al. J. Vis. Exp. 2011; (50): 2685.
Optic Nerve Crush Use in Your Research Program
Optic nerve crush allows for the evaluation of drug intervention following neuronal injury at the cellular and biochemical levels. Immunostaining can monitor therapeutic effects, and immunoassays can be developed to track biomarkers following treatment.
Download Brochure
Bioanalytical
Our bioanalytical services goal is to meet your project schedules and providing the highest quality data to facilitate decisions concerning your drug candidate. Our scientists are skilled in method development, validation, and sample analysis of small and large molecular weight compounds, metabolites, and peptides in biological matrices using liquid chromatography with tandem mass spectrometry (LC-MS/MS). We have three different LC/MS platforms (SCIEX 5000, SCIEX 4000, Thermo TSQ Quantum Ultra). Analyses can be conducted following FDA GLP guidelines, as needed.
What we can do for you:
- rapid screening bioanalytical methods
- method development, validation, and sample analysis following FDA GLP and OECD guidances
- preclinical pharmacokinetics
- PK/PD assessment
- toxicokinetics
- bioavailability/bioequivalence
- Ocular, prodrug and metabolite PK
Biochemistry
PharmOptima staff has provided biochemistry services to companies world-wide. We specialize in developing custom assays to meet specific client research needs.
What we can do for you:
- cell-based assays
- biomarker assays
- ELISA
- MSD electrochemiluminescence
- ligand binding assays
- Protein purification and characterization:
- Affinity protein purification methods limited to:
- His-tag
- GST-tag
- MYC-tag
- FLAG-tag
- Fc fusion proteins
- Antibodies via protein A/G
- Traditional methods
- Affinity protein purification methods limited to:
- antibody purification and labeling (eg Biotin and sulfo-tag ruthenium for ECL applications)
- stable mammalian cell line generation and characterization
- protein expression
- eukaryotic expression systems (mammalian cell and insect cell/baculovirus)
We develop biomarkers and assays.
Utilizing MSD and ELISA Technology*, biomarkers are available from human, rat, mouse and non-human primate in many cases. A few examples include:
- metabolic markers
- oncology markers
- vascular markers
- cytokines and chemokines
- cell signaling pathways
- Alzheimer’s (Aβ38, Aβ40, Aβ42 and Tau, Total Tau)
- kidney injury
- cardiac and muscle
- liver injury
- inflammation
We provide custom assay development services to customers worldwide. PharmOptima’s scientists can work with you to develop assays to meet your specific research needs. Our scientists have extensive experience in the different custom assay development services listed below.
- Immunosorbant assays, traditional ELISA and ECL
- Development of multiplex immunoassays using MesoScale Discovery technology
- Cell-based assays: signal transduction (eg cAMP)
- Ligand binding assays
- Enzyme assays and kinetics
- Inhibitor mechanism and validation
- MSD Certification such as Human Aß42 and Human Total Tau
Nuclease Protection Assays
What is RNA analysis by nuclease protection?
A nuclease protection assay is a laboratory technique used in biochemistry and genetics to identify individual RNA molecules in a heterogeneous RNA sample extracted from cells. The technique can identify one or more RNA molecules of known sequence even at low total concentration.
Oligonucleotides are traditionally used in polymerase chain reaction applications for in vitro gene amplification. However, recently, oligonucleotides are being evaluated for therapeutic applications either by increasing gene expression via splicing intervention (e.g., Nusinersin in Spinal Muscular Atrophy treatment), decreasing gene expression using anti-sense sequences to inhibit transcription or translation (e.g., Mipomersin inhibition of apoB-100 translation in the treatment of Familial Hypercholesterolemia) or in the case of aptamers, inhibiting protein activity (e.g., Pegaptinib inhibition of vascular endothelial growth factor, VEGF, in the treatment of macular degeneration).
Pharmacokinetic studies for oligonucleotide drugs require specific assays. One assay format that has been put to use by PharmOptima’s scientists is the nuclease protection assay (Figure 1). The nuclease protection assay (see Figure) uses a labeled nucleotide sequence complementary to the oligonucleotide drug to capture free drug from solution. Following capture, the binding reaction is treated with S1 single stranded nuclease to remove non-bound capture oligonucleotide, removing non-specific signal.
PharmOptima uses Meso Scale Discovery (MSD) technology for this assay. The MSD platform, while similar to ELISA, uses electrochemiluminescence detection to analyze complex sample matrices for a variety of analytes.
Assay Characteristics:
- Sensitivity pg/mL
- Large dynamic range pg/mL through microgram/mL
- Assay reproducibility
- Quick turn around time (2-3 hours) once assay is established
- Minimal matrix effects
The nuclease protection assay uses a labeled nucleotide sequence complementary to the oligonucleotide drug to capture the oligonucleotide drug from solution forming a double stranded sequence. S1 nuclease is an enzyme that degrades single stranded DNA and RNA. In the nuclease protection assay, following the capture step, S1 nuclease is used to degrade any non-double stranded nucleic acid leaving the double stranded captured drug “protected”. Signal from the double stranded protected sequence is then used for detection and quantification of the oligonucleotide drug.
In vivo
Your compounds can be tested in PharmOptima’s vivarium for in vivo bioavailability, metabolism, pharmacologic efficacy, and tolerance.
What we can do for you:
- extensive ophthalmic expertise
- transgenic mouse colony capabilities
- evaluation of drug tolerance, integration of studies with drug delivery and pharmacokinetics (PK)
Routes of Administration
- Oral Gavage
- Subcutaneous
- Intravenous/Infusion
- Intact Spinal Cord Collection
- Intraperitoneal
- Intramuscular
- Intravitreal
- Topical (Ocular)
- Dermal
- Intratracheal
- Intranasal Inhalation/Instillation
- Intrathecal
Ocular Tissue Collection
Anterior
- Aqueous humor, Cornea, Conjunctiva (palpebral and/or bulbar)
- Meibomian Glands
- Trabecular meshwork, Iris-Ciliary body, Lens
Posterior
- Vitreous humor, Optic Nerve
- Sclera, Choroid (central and/or peripheral)
- Retina (central and/or peripheral)
Ocular Tissue Collection
Anterior
- Aqueous humor, Cornea, Conjunctiva (palpebral and/or bulbar)
- Meibomian Glands
- Trabecular meshwork, Iris-Ciliary body, Lens
Posterior
- Vitreous humor, Optic Nerve
- Sclera, Choroid (central and/or peripheral)
- Retina (central and/or peripheral)
SMN Assay Development
Our Spinal Muscular Atrophy (SMA) services begin with research and concludes with a high level of technical expertise.
GD3 researchers have worked with organizations such as the Spinal Muscular Atrophy Foundation, universities and pharmaceutical companies on critical SMA research. SMA, the leading genetic cause of infant death, is caused by defects in the Survival Motor Neuron 1 (SMN1) gene that encodes SMN protein. SMA patients have at least one copy of a similar gene (SMN2) that produces SMN protein, although in reduced amounts. Infantile-onset spinal muscular atrophy is the most common genetic cause of infant mortality, typically resulting in death preceding age two. Low-level production of survival motor neuron protein (SMN) results in a loss of specialized nerve cells called motor neurons that control muscle movement.
In recent years, new SMN-enhancing therapeutics have been developed. Since limited knowledge of baseline and drug-induced SMN levels in disease-relevant tissues hinders efforts to optimize these treatments, clinical trials in this population require understanding disease progression and identifying meaningful biomarkers to hasten therapeutic development and predict outcomes. Clinical studies require a readily accessible means of tracking SMN levels in the patient. GD3 scientists have developed an immunoassay system that monitors SMN protein levels in whole blood. The assay can detect and quantify SMN protein from as little as 5 microliters of whole blood, which can be obtained from a finger prick.
GD3 experts have supported research in published studies of spinal muscular atrophy, including:
- Whole blood survival motor neuron protein levels correlate with severity of denervation in spinal muscular atrophy
- Natural history of infantile-onset spinal muscular atrophy
- Age-dependent SMN expression in disease-relevant tissue and implications for SMA treatment
- Mild SMN missense alleles are only functional in the presence of SMN2 in mammals
- Evaluation of potential effects of Plastin 3 overexpression and low-dose SMN-antisense oligonucleotides on putative biomarkers in spinal muscular atrophy mice (plastin 3, also known as T-plastin or fimbrin)
- Normalization of Patient-Identified Plasma Biomarkers in SMN2 Mice following Postnatal SMN Restoration
What we can do for you:
- Novel Method Development and Standard use:
- Immuno Assays
- Immunogenicity Assays
- Cell-based Assays
- Nuclease Protection Assays
- Ligand Binding Assays
- Oligonucleotide Assays
- ELISA Assays
- SMA transgenic mouse colony capabilities
- LC-Mass Spec analysis of small molecules
Let GD3 collaborative biology/bioanalytical teams generate high-quality leads to advance your programs.
