# Presentation: 941529

## A MINOR USE ANIMAL DRUG PROGRAM (NRSP-7)MUMS RESEARCHSpecies Grouping StrategiesDr. John G. BabishNRSP-7 National Coordinator

**A NATIONAL AGRICULTURAL PROGRAM **
**TO APPROVE ANIMAL DRUGS **
**FOR MINOR SPECIES AND USES**

## Economics of Species Grouping

- 	Agricultural production of fish, game birds, sheep, goats, ratites bees and deer in the United States is critically important to numerous regional economies in the United States.  This diverse aggregation of minor species represents approximately $1.5 billion in state and local US farm revenues annually.  Additionally, processing and export of minor species food and fiber products represents an additional $4.5 billion of revenue.  Individually, however, these minor species represent drug markets too small to provide a sufficient return on the high cost of developing a new drug application.

## Economics of Species Grouping

- New animal drug approval ~ $20 million (8 - 10 years)
- Addition of a new species to the label ~ $2 - 8 million
- NRSP-7 cost per species = $0.3 million
- Currently there are 13 active NRSP-7 projects
- and 41 potential projects
- Cost to industry for these approvals ~ $108 - 432 million (USD)
- Cost to NRSP-7 ~ $16 million (USD)

## Species per Class

- Number Represented

- Cost effectiveness of species grouping is greatest in the aquatic and avian groups.

## Approaches to Species Grouping

**Characterizatizing and clustering of all metabolites of representative drugs in species of interest.**
**(Metabolomics)**
**Characterizing and clustering the xenobiotic biotransformation capacity in the species of interest**
**(Genomics, proteomics and model metabolomics)**** **

## Gene Expression and Protein Function

**DNA**

**Primary**
**RNA**
**Transcript**

**mRNA**

**mRNA Degradation**

**Primary**
**Protein Product**

**Finished**
**Protein Product**

**Active**
**Protein**

**Genomics**

**Proteomics**

**Translational**
**Control**

**Transport**
**Control**

**Processing**
**Control**

**Control**

**Transcriptional**

**Proteolysis Control**

**Proteolytic Processing**

** ****Chemical Modification**
**Complex Formation**

**Metabolomics**

**Metabolites**

**Notes:**
	Those factors that affect drug activity include (1) mechanism of action, (2) rate and extent of absorption, (3) extent of distribution, (4) qualitative and  quantitative rates of metabolism or biotransformation, and (5) rate of excretion.  

	Both genetic and environmental factors can greatly influence (2) through (5).  Structure  is the primary factor involved in the determination of mechanism of action.  

## Species GroupingDeterminants of Drug Activity

**Mechanism of action**
**Absorption**
**Distribution**
**Metabolism or biotransformation **
**Excretion**

**Notes:**
	Those factors that affect drug activity include (1) mechanism of action, (2) rate and extent of absorption, (3) extent of distribution, (4) qualitative and  quantitative rates of metabolism or biotransformation, and (5) rate of excretion.  

	Both genetic and environmental factors can greatly influence (2) through (5).  Structure  is the primary factor involved in the determination of mechanism of action.  

## Determinants of Drug Activity

**Notes:**
	Here we have a schematic depiction of the factors related to the five determinants of drug activity.  For the purposes of this presentation, we will be focusing on biotransformation systems in the Phase I or oxidative group of reactions.  Within the  Phase I group of reactions, we will deal specifically with the cytochrome P-450 or CYP superfamily of enzymes.  

## Drug Biotransformation by P-450 Isozymes

**CYP3A**

**CYP2D6**

**CYP2C**

**CYP1A2**

**CYP2E1**

**Notes:**
	The pie chart above depicts the relative contribution of five CYP isozymes representing three families of isozymes.  As can be readily seen, the CYP3A subfamily accounts for more than 50% of the drug biotransformation reactions.  While quantitatively small, the CYP2E1 subfamily is important due to its inducibility by ethanol and metabolic activation of acetaminophen to reactive metabolites.  

## CYP Families and SubfamiliesGeneral Substrate Requirements

**1A2**

**2B6**

**2C19**

**2D6**

**3A4,5,7**

**2E1**

**2C9**

**2A6**

**1A1**

**-Low MW with diverse structures**

**Plannar polyaromatic**

**High  MW with diverse structures**

**-Basic compounds**
**With N-**

**-Non-planner including amides**

**-Non-planner, V-shaped structures**

**Notes:**
	The P450 enzymes are a large superfamily of hemoprotein monooxygenases in prokaryotes and eukaryotes.  These enzymes play an important role in the oxidative metabolism of a wide variety of both exogenous and endogenous substrates.  At present, more than 230 distinct P450 genes and pseudogenes have been identified.  
	This superfamily comprises 36 gene families, of which 10 include subfamilies and 26 are represented by only one gene.  In addition to the amino acid sequence homology, gene and  genome structure data were used to model their evolution.  
	The eukaryotic P450 families always form stable clusters such as {CYP1, CYP2, CYP17, CYP21}.  {CYP3, CYP4, CYP6, CYP52, CYP102}, AND {CYP11, CYP27}.  CYP2, the most expansive P450 family, can be divided into five clusters of subfamilies: {CYP2A, CYP2B, CYP2G}, {CYP2C, CYP2E, CYP2H}, {CYP2D}, {CYP2F}, and {CYP2J}.   The trout CYP1A1 always forms the most outlying b ranch in its family.  
	The broad classification of substrate specificity is listed for the 1A, 2B, 2C, 2E and 3A subfamilies that will be the focus of the presentation.  There is some degree of overlapping substrate specificity,  with CPY3A4 having an extremely wide range of diversity in substrate structures.  CPY3A4 is often referred to as the “garbage disposal” of the CYP isozymes.   

## Species Grouping Study DesignAquaculture 

**Characterizing CYP by Western Blotting and Enzymatic Analysis**

** ****Rainbow trout,**
** ****Tilapia,**
** ****Walleye, **
** ****Yellow perch,**
** ****Summer flounder,**
** ****Hybrid striped bass,**
** ****Channel catfish **

**Initial work demonstrates that antibodies to human CYP, CYP1A1 and CYP3A, cross-react with several aquatic species.**

## Species Grouping EffortAquaculture

**Observational Study of Hepatic CYP1A2 and CYP3A4 in Hybrid Striped Bass, Summer, Channel Catfish and Tilapia **

-  Fifty fish per study;
-  Fish were treated with 82.8 mg OTC/kg body weight for ten days;
-   Ten fish were sampled at pre-treatment and post-treatment days 1, 6, 10 and 21.
-   Body weights, liver weights, and hepatic CYP1A2 and CYP3A4 activities were monitored pre-dosing and at D1, D6, D10 and D21.

## Hepatic CYP1A2 and CYP3A4 Activity in Hybrid Striped Bass Following Oxytetracycline Treatment for Ten Days

**No differences were seen in CYP1A2 or CYP3A4 activity during the post-treatment observational period.**

**CEC = CYP1A2**
**BFC = CYP3A4**
**BzRes = CYP3A4**

## Hepatic CYP1A2 and CYP3A4 Activity in Channel Catfish Following Oxytetracycline Treatment for Ten Days  

**.  **

**CEC = CYP1A2**
**BFC = CYP3A4**
**BzRes = CYP3A4**

**No differences were seen in CYP1A2 during the post-treatment observational period.  CYP3A4 was increased from pre-treatment at D1, but returned to pre-treatment levels at D6, D11 and D21 as measured by BzRes.  With BFC as substrate  there were increases in CYP3A4 activity at D11 and D21.  **

## Hepatic CYP1A2 and CYP3A4 Activity in Immature Tilapia Following Oxytetracycline Treatment for Ten Days  

**.  **

**CEC = CYP1A2**
**BFC = CYP3A4**
**BzRes = CYP3A4**

**CYP1A2 activity decreased during the post-treatment observational period.  CYP3A4 decreased at D6 and D11 with BzRes and did not change with BFC as substrate.  **

## CYP1A2 Immunoblot in Hybrid Striped Bass

## CYP3A Immunoblot in Hybrid Striped Bass

## Species Grouping Development Platform

- Gene Expression

- Proteomics

** ****Transcriptome**
**      **Biological process
** ****Proteome**
**      **Protein interactions
** ****Metabolome**
-       Metabolites** **
**Functional integration **
**      **Pathways
** ****Clustering of information**
**      **Groping of species

** ****Gene annotation**
** ****Protein annotation**
** ****Protein interactions**
** ****Pathway membership**
** ****Integrated biological organization**
** ****Literature**

**Animal Tissue Bank**

**Database**

## Conclusions

**     ****Species grouping through characterization and clustering of biotransformation capacity (proteome and model metabolome) can be more rapid and cost effective than drug-metabolome characterization.  **
**     ****Model substrates and inhibitors for CYP characterization are well-developed and extensively validated.**
**     ****Antibodies for quantification of CYP protein expression are not well characterized and present a limiting factor in biotransformation proteome characterization.**
**    ****Primers for characterizing mRNA and the biotransformation genome are developed for murine and human genomes.  Addition of genomic characterization of the biotransformation capacity could greatly improve the quantification and clustering of species grouping.    **

## Associates

**   ****Dr. Paul R. Bowser**
**   ****Dr. Art Craigmill**
**  ****Dr. Natalija Topic Popovic**
**  ****Mr. Greg Wooster**