Case 1: Hydrolysis Study with Unlabelled Insoluble Compound at very Low Concentration Levels
- For such compounds, radiollabeled material is typically used due to limitations of assay sensitivity.
- Compound had negligible water solubility and radiollabeled compound was not available.
- A method capable of following the degradation products was required (LC-MS/MS can readily follow the disappearance of parent compound, but not production of hydrolysis products).
- The team developed a highly sensitive HPLC analytical method, including concentrating the samples and reconstituting in a small volume. This method could monitor of up to 90% degradation of parent, and of degradation products more than 5%.
- The method was successfully employed to complete the hydrolysis study with cold compound. The 3 major hydrolysis products formed during the reaction were identified and quantified.
Case 2: Impurity Profiling and Five-Batch Analysis of Agrochemical Technical Material
- A total of 9 impurities, including 6 unknown impurities were found during screening prior to a GLP 5-batch analysis of agrochemical technical material. Structural elucidation of these impurities was essential to generate their reference standards.
- Mass spectra (MS) and MS/MS of each impurity were recorded, and based on the synthetic process, structures of 3 of the unknown impurities were proposed and confirmed.
- Standards for 2 of these impurities were obtained commercially, and the third was synthesized.
- The remaining 3 unidentified impurities were isolated from the technical material using prep-HPLC following its enrichment for these impurities.
- After characterization, these impurities were used for successful completion of the GLP 5-batch analysis study.
Case 3: Dose Formulation Analytical Method Development
- Test item was a mixture of several compounds and due to the nature of these compounds (no chromophores and non-volatile), no routine methods could be used for dose formulation analysis.
- A novel approach for dose formulation analysis was followed. As per this new approach, the dose formulation analysis was done based on the total nitrogen estimation by Kjeldahl method (amount of total nitrogen in the test item as such and total nitrogen in the dose formulations were analyzed).
- All the Safety assessment studies could be completed with the support of this dose formulation analysis approach.
- Sponsor was very happy with this approach.
Case 1: Novel Sample Processing Approach to Overcome Formulation Effects on LC-MS/MS Bioanalytical Results
- Plasma LC-MS/MS analysis for the active sphingolipid after IV administration of a sphingolipid liposomal formulation showed very short half-life when compared to a previous study done with radiolabeled lipid formulation.
- The initial plasma bioanalytical method using ACN protein precipitation could successfully quantify sphingolipid after spiking liposome formulation in-vitro in plasma.
- When applied to study samples, measured concentrations at early time points (5 to 15 min post dose) were similar to those seen in a radiolabel liposome study. However, the terminal half-life was substantially smaller with the LC-MS/MS data.
- Detailed experiments including variety of sample processing techniques revealed that sample preparation plays a significant role in the actual recovery of analytes from in-vivo samples.
- When the same study samples were prepared under unusually strong conditions (45°C for 8 h in an oven) to de-aggregate the constituent lipids in the liposome, the PK of the sphingolipd was similar to that seen in the radiolabeled study. This may result from complexation of the liposomal lipid after dosing in vivo.
This case study was presented as poster in Applied Pharmaceutical Analysis (APA) India 2015 and was awarded a prize.
Case 2: A Validated Method to Quantify Multiple Analytes Including 2 Prodrugs and their Metabolites in Free and Bound Form in Plasma following Administration of a Fixed Dose Combination of Nutraceuticals
- For a fixed dose combination product of nutraceuticals, to quantitate in plasma two ethyl ester prodrugs, as well as free and triglyceride-bound concentrations of their metabolites. The metabolites were endogenous bioactive lipids. This required quantification of 6 analytes in the sample.
- Esterase inhibitor was added to blood samples at the time of collection to quench further metabolism. The 2 ester prodrugs were quantitated by LC-MS/MS method.
- A separate LC-MS/MS bioanalytical method was developed to simultaneously quantify the 2 major metabolites.
- Free and triglyceride-bound levels of the metabolites were determined by processing each plasma sample by 2 different methods: (A) normal liquid-liquid extraction to measure free metabolites level and (B) acidic and alkali digestion followed by liquid-liquid extraction to measure total metabolites levels.
- Because the metabolites were endogenous bioactive lipids, various surrogate matrices were evaluated for use in paring the calibration curve and QC samples.
- This method was validated as per FDA requirements and applied to GLP studies. The studies were inspected by the USFDA and met the inspectional standards.
Case 3: A Bioanalytical Method for Parent Vasodilator Drug and its 2 Active Positional Isomers Metabolites in Plasma to meet Recent FDA Guideline Requirements
- Quantification of the active positional isomers was required at very low levels as per a recent Office of Generic Drugs, USFDA guidance (2015). There is no literature precedence for this method.
- The active positional isomers (metabolites) in the plasma samples were chromatographically separated on a chiral column – this was a key challenge which was overcome.
- A second challenge was optimization of the sample processing and the mass spec conditions to achieve the desired sensitivity – this was addressed following multiple simultaneous efforts during method development.
- This approach was successfully applied to clinical study samples.
Case 4: GLP Bioanalysis - Large pharma ran a Blinded Bioanalytical Trial
- Sensitivity of 50 pg/mL with 10,000 fold dynamic range
- No structural or physicochemical information on the compound provided
- Advinus developed and validated a suitable highly sensitive method after identifying that a sodium adduct was highly sensitive
- Validated the method with two overlapping CCs
- The method was applied to mouse, rat & dog plasma samples from IND-enabling GLP toxicology studies
- Four full INDs were completed for the client; became bioanalytical partner for Phase I clinical trials in US
Case 5: Clinical Bioanalysis support for US based Industry Leader
- Trials were managed by a US based CRO; study performed in continental US
- Sensitive method was developed suitable to the low doses in the initial cohorts
- All logistics were worked out. Samples were received at Advinus within 72 hours of sample collection
- Data reported within 72 hours to enable exposure-guided dose escalation. Interim PK calculations also provided with the bioanalytical data
Case 1: Discovery DMPK Support for Active Lead Optimization Programs for Large Pharma
- To qualify as the preferred service provider in a head-to-head proficiency assessment as compared to other service providers.
- To set up and validate the in-vitro CYP inhibition and protein binding assays to the Large Pharma’s desired level of detail/requirement.
- Advinus developed and validated all the in vitro assays.
- Confirmed assay validation using Sponsor’s blinded compounds, and followed a screening cascade for the program compounds.
- Tier 1: Solubility, Microsomal Metabolic Stability, Caco-2 Permeability
- Tier 2: CYP Inhibition in HLM by LC-MS/MS, Plasma Protein Binding and Microsomal Binding
- Tier 3: Bioavailability studies in rat and dog
- Collaborated with Sponsor to devise a definitive PPB equilibrium dialysis assay using LC-MS/MS based quantification (Zamek-Gliszczynski MJ et al. Validation of 96-well equilibrium dialysis with non-radiolabeled drug for definitive measurement of protein binding and application to clinical development of highly-bound drugs. J. Pharm. Sci. 2011 Jun: 100(6):2498-507).
- All shipping and project management processes were worked out.
- Advinus turned around data within 1 week of receiving compounds to enable sponsor to quickly decide on moving compound along the screening cascade.
Case 2: Identifying a Metabolic Soft Spot and its Effect on Activity
- To assess if metabolism would affect the activity of an NCE – that exhibits a de-methylated metabolite and possess two possible sites of de-methylation.
- Established the site of metabolism using LCMS/MS fragmentation patterns of the parent and the metabolite and confirmed that metabolism occurred on a substituent distant from the pharmacophore not affecting the activity of the molecule.
Case 3: Ocular Dose Formulation Preparation
- To prepare a sterile ocular dose formulation in-house for pre-clinical GLP toxicology testing.
- Prepared an in-house dose formulation that meets the standard specifications for ocular preparation like assay, sterility and bacterial endotoxins.
Case 4: Validation of CYP Inhibition Study in Human Hepatocytes
- To determine if a compound showing CYP inhibition potential in-vitro in microsomes but low uptake into hepatocytes, shows CYP inhibition liability in hepatocytes.
- Advinus validated CYP inhibition assay in hepatocytes.
- Established that the compound did not inhibit any of the major CYPs in the hepatocyte preparation, suggesting a reduced liability for in vivo drug-drug interactions.
Case 5: Species Differences in Metabolism and its Influence on Tox Findings
- To understand the reason for enhanced activity/toxicity of a compound in dogs compared to rats.
- Investigation using mass spectrometry revealed presence of a previously unknown species specific metabolite in dogs.
- Established that the metabolite was formed through acetyl transfer by N-acetyl transferases that are absent in dogs.
Case 1: Performance of Juvenile Toxicology Studies to Support NDA for Pediatric Population
- This study required several additional factors to be considered including:
- Concept of timed pregnancy and parturition for pool of juvenile rats.
- Equitable distribution of offspring to all groups from each dam.
- Dosing from very young age in rats (Day 10).
- Selection of appropriate dosing method and sample collection.
- No stress, gentle dosing, handling and zero errors.
- The highly experienced team at Advinus was able to perform this study and it was successfully accepted by US regulators. This paved the way for several subsequent studies from the sponsor.
Case 2: Establishment and Standardization of Standard for Exchange of Nonclinical Data (SEND) Format
- The USFDA has recently mandated that preclinical or nonclinical data for studies beginning after December 17, 2016 should be provided in SEND format.
- Advinus established in 2015 itself for the first time in India capability for SEND formatting of data. It is one among few facilities in the world with this capability.
- Advinus has successfully enabled submission of multiple datasets by sponsor to USFDA from 26 week Tg.rasH2 study and 2 year carcinogenicity study.
Case 3: Conduct of 2 year Carcinogenicity Study of a Test Compound having a Short Half-life
- The short half-life in rodents prevented the achievement of adequate exposures to enable a safety margin.
- The team performed twice-daily dosing of 500 animals for 730 days to overcome the issue of short half-life and maximize the possible exposure.
- There were zero dosing errors and no stress-related incidents, and the survival rate was higher than that reported in the literature.
- This type of very long duration study required exemplary concentration, diligence, dedication, motivation of the staff which they showed.
- It represented a rare feat in the 25 years history of Advinus; few labs in the world have achieved this.
Case 4: Conduct of Shorter Duration Carcinogenicity Study using Transgenic rasH2 Mice
- USFDA has recently approved substitution of the traditional 2 year (78 weeks) rodent carci study with a 6 month (26 weeks) study in the rasH2 transgenic mice. This is based on the propensity of these knock-out mice to develop tumors earlier in their lifespan, thus shortening the overall duration of the study.
- The team developed systems and processes to handle sensitive transgenic mice
- There was no undue mortality and morbidity, and the positive control data developed for MNU (N-Nitroso-N-Methylurea) and urethane met the validation requirement for development of neoplastic changes. The report was successfully submitted.
This represents a rare achievement in preclinical tox testing – only a handful of labs in the world have experience in performing this type study.
Case 1: Process Development of a Chiral NCE with High Synthetic Complexity
- Original process had 13 steps, was expensive and used hazardous raw materials unsuitable for larger scale.
- There was a tendency for racemization in the penultimate step at a critical chiral center.
- Chromatographic purification was involved in almost every stage, with final API isolation by preparative HPLC.
- The challenge was to develop a safe, inexpensive, process-friendly route, without column purifications and developing conditions that avoided racemization.
- A novel process was designed with inexpensive locally available raw materials.
- The cause of racemization was identified and addressed after testing various chiral reagents.
- Column chromatographic purifications were replaced by crystallizations at all the steps.
- Green chemistry principles were applied to minimize the solvent(s) and reagent(s) usage.
- Optimized the process to synthesize API having >99% chemical and >97% chiral purity.
- Delivered multi kilogram quantities of the API on time to support the pre-clinical and clinical tox studies.
Case 2: Process development of agrochemical ingredient for a leading Indian company
- To develop an innovative, non-infringing, scalable, cost-effective, sustainable process with zero toxic waste for an important crop-protection chemical.
- The company wanted to file 9(3) registration of this agrochemical for exclusive manufacturing rights.
- Designed and demonstrated 2 non-infringing synthetic routes for A. I.
- Developed a cost-effective process using locally available raw materials and safe operations.
- Optimized the process to manufacture on commercial scale.
- No use of catalysts, reagents involving heavy metals or toxic solvents thus achieving zero discharge of toxic waste in environment.
Case 3: Development and scale-up of an investigational new drug molecule for a European research foundation
- The client’s medicinal chemistry scheme had multiple chromatography purifications, separation of the required isomer using chiral HPLC, and poor yield.
- To develop a novel, scalable, cost-effective, stereo-selective process with high atom economy to produce API for pre-clinical and early clinical studies.
- Stereo selectivity was achieved using Sharpless epoxidation.
- Telescopic process was employed in several steps.
- Demonstrated process on 10.0 kg (non-GMP) and 4.0 kg (GMP) scale with chiral purity of 99 % ee.
- Developed a recrystallization method to achieve desired particle size.
- Process development involved selective debromination.
- Results published in OPRD.
Case 1: Liver-Directed Glucokinase Modulators for T2D
Liver-Directed Glucokinase Modulators for T2D: Unique Way to Retain Efficacy and Eliminate the Risk of Hypoglycemia
- The biology concept was novel. Pharmacological PoC was also unprecedented.
- The complete discovery phase had to be built on in-house hypothesis and new SAR development.
- Biology experts hypothesized that liver-directed Glucokinase (GK) modulation, while sparing the pancreas, will exert an antihyperglycemic effect without causing hypoglycemia.
- Medicinal chemistry experts hypothesized that certain structural motifs will distribute a compound selectively to the liver, compared to systemic circulation and other tissues.
- In addition, we postulated that moderately potent activators would activate GK in the liver due to high local concentrations, but be unable to activate pancreas GK even at higher dose, providing a wide therapeutic window.
- Combination of the above three unique hypotheses, led to the successful discovery of GKM-001, a leading second generation Glucokinase modulator with proven translation of efficacy from preclinical to human, and devoid of any risk of hypoglycemia.
- Successful completion of Phase-Ib clinical trials. Preparing for Phase-II.
Case 2: A Fast-Follow-up Program for T2D and Obesity
New Hypothesis and well Differentiated Compound in a Fast-Follow-up Program for T2D and Obesity
- The leading competitor was selective for only one isoform of the target. This limited its efficacy. Available medicinal chemistry as well as pharmacology literature also described only a single isoform targeting approach.
- There were also some potential safety concerns with the leading compounds.
- A new medicinal chemistry plan had to be developed that mitigated these potential safety concerns and at the same time provided a clear differentiation strategy our competitors.
- A proprietary series was developed, yielding compounds with varying selectivity over a second isoform of the target, also hypothesized to provide an advantage over the competition for T2D and obesity.
- Detailed pharmacology profiling of four optimized leads with varying selectivity, ultimately led us to select one clinical candidate that engaged both isoforms of the target and showed superior efficacy over the leading competitor.
- Advinus’ unique approach successfully delivered a well differentiated clinical candidate for further development for T2D and obesity.
- As a fast-follow-on program, candidate selection was done in 2 years, including the time taken for establishing an unprecedented SAR and differentiation strategy.
- Completion of Phase-II clinical trials.
Case 3: Candidate Nomination in Record Time
- Fast follow on: competitors in phase I/II
- Therapeutic indication: Identify new indication(s) other than competitors and/or differentiation over current indication in clinic
- Identify novel hit for analoging in absence of HTS screen
- Deliver CCN in two years
- Novel and diverse six chemotypes designed to carry out parallel SAR to identify HITS in first three months
- Identified four HITS and prioritized two series to provide two LEADS within 6 months
- Developed novel in-vivo PD Assay
- Nominated Optimized Lead for Candidate Selection within 10 months,
- Progress was halted due to phototoxicity and human hepatocyte instability
- Second lead series showed CYP TDI liability
- SAR knowledge from earlier two series helped to design a new chemotype (Third) to overcome above hurdles in time
- Identified new indication by exploring efficacy in various disease models and data mining with client’s help
- Identified clinical candidate with excellent efficacy and good therapeutic window and potential back-up compound is being profiled
- Filed three chemical composition of matter patents
- This programs achieved clinical candidate nomination milestone from collaborator
- Nominated clinical candidate is being profiled for new indication and IND enabling studies ongoing.