Progress 06/01/14 to 07/31/15
Outputs
Target Audience:The University of Missouri College of Business Administration has a MBA course in Entrepreneurship. As part of the course,a student group was assigned to do a business planfor the production animal markety of the antimicrobial and weight enhancing peptidesdeveloped in this SBIR Phase I grant. The students are actually contacting and interviewing various organizations/companies involved in the steer, pig, and poultry industries. As part of their questions aboput the market need for our potential pharmaceuticval products,our target audiences are learning about our company and its products. as part of their Changes/Problems:As noted on a previous page, the FDA has a new position on approval of antimicrobials for enhancing weight gain in production animals; such drugs will not be approved. Thus, we need to develop separate drugs for antimicrobial actions and for enhanced feed efficiency/weight gain. What opportunities for training and professional development has the project provided?We had both an undergraduate and a veterinary student working on our SBIR Phase I project. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
While we initially were planning to develop a peptide drug that combined antimicrobial and weight enhancing activities, recent position statements from the FDA caused a change in our business plan. FDA decided to not allow the approval/sale of antimicrobials for weight -enhancing actions in production animals, due to the possibility of the development of microbial resistance. While we can provide theoretical functional arguements against this concept, our development workon ahuman antimicrobial for hospital-associated infections (there is significant DOD interest in our work) will provide FDA with further support for their position. Thus, we decided to develop separate therapeutics for each problem, with separate business plans.
Publications
|
Progress 06/01/14 to 05/31/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? Completion of project goals.
Impacts
What was accomplished under these goals?
Development of Anti-Microbial Peptides (AMPs) We identified anti-microbial activity in several analogs of our melanocortin antagonist therapeutic (TCMCB07, Fig 1 below), by testing their effects on both Gram positive and Gram negative microbes (Table 1, below). These AMPs had cationic residue additions, and substitutions of aromatic residues with naphthylalanine (Nal; a synthetic tryptophan analog), since both of these derivatizations enhance antimicrobial activity (33). Our first 5 most effective AMP peptides caused substantial cell agglutination effects, and 4 of these have additional effects on cell division processes, inhibition of division. The ability of an AMP to cause agglutination of bacterial cells is a beneficial attribute, as this could lead to increased phagocytosis and clearance of bacteria by macrophages and neutrophils (34). Several previously unreported aspects of AMPs are seen in the tables below. The C-terminal dipeptide sequence (Table 1, Val-Pro vs. Val-Phe or Phe-Val) appears to direct relative antimicrobial activity towards Gram-negative or Gram-positive species. Another aspect of antimicrobial peptides is seen in the two control peptides in Table 1; melittin and Mas7. Both are cationic-nonpolar proteins, similar to AMPs. Melittin is an antimicrobial peptide derived from bee venom, while Mas7 is a structural analogue of mastoparan and a known activator of cell membrane heterotrimeric Gi-proteins and its downstream effectors. Both showed significant differential antimicrobial activity; significantly better towards Gram-negative organisms. Fig. 1: TCMCB07; Orally active melanocortin 4 receptor antagonist Ac-Nle-c[Asp-Pro-d-Nal(2′)-Arg-Trp-Lys]-d-Val-d-Pro-NH2 Development of an AMP with Anti-Biofilm Activity We designed a hybrid anti-biofilm-antimicrobial peptide drug (AB01; Fig 2, below), using our platform technology, that incorporates a potential melanocortin receptor antagonist sequence. The C-terminal di-peptide sequence of AB01 (dVal-dPro) directed relative antimicrobial activity towards Gram-positive bacterial species (see Table 1, above). We are currently assessing the anti-microbial and anti-biofilm properties of AB01 in both Gram-positive and negative species, with comparisons to a previously published linear AMP (Model 01) and a cyclized analog of Model 01 (AM11); see Fig. 2. Fig. 2 Model 01, an AMP of the (RW)n series (RW)4: (41): Ac-RWRWRWRW-conh2 AM11, a cyclized analog of the (RW)n series c(RW)3: Ac-Nle-c[Asp-RWRWRW-Lys]-dVal-dPro-conh2 AB01, a hybrid AMP/anti-biofilm peptide: Ac-FRIRVRV-c[Asp-RR-dNal(2')-FWR-Lys]-dVal-dPro-conh2 The initial results from these experiments (Fig 3A-F, below) clearly demonstrate the superior anti-microbial actions of AB01 (vs. Model 01 and AM11) against a model Gram-positive mycobacteria, M smegmatis, before and after biofilm production. We compared growth inhibition of cells attached to a plate surface (pre-biofilm formation), and for 1 and 2 days after attachment [after the biofilm developed (Figs 3A-C)]. These experiments were performed in 96 well plates, in which the control culture's attached growth rate was measured in the absence of an AMP. Attached cell growth and biofilm production was measured by crystal violet staining, with quantification in a plate reader at an absorbance of 561 nm (42). Control attached bacterial growth was designated as 100%, and the effect of added AMPs on the attached cell growth rate was quantified as a percent of the control growth (greater or lesser). The MIC (minimum inhibitory concentration for 100% suppression of attached cell growth) of AB01 was about 2-3µM during the initial growth phase (before a mature biofilm was secreted; Fig 3A), with an MIC of about 10µM against a mature biofilm (Figs 3B&C). Of the other two AMPs, Model 01 had the best activity; with an MIC of about 10µM in the M smegmatis biofilm growth phase (Fig. 3A), and >100µM after the biofilm was established (Figs. 3B&C).. Figs. 3D-F show the effects of the three AMPs on E. coli attached cell growth; initially (3D) and as a biofilm developed (3E&F). The initial MIC of AB01 was 100 µM; about 10-100-fold less effective than against a Gram-positive species (Fig 3A). This is consistent with the specificity data for different C-terminal dipeptides depicted in Table #1, since AB01 has a dipeptide relatively specific for Gram-positive bacteria. The two other AMPs showed some inhibition of growth at the early phase of bacterial cell attachment (Fig. 3D). All three AMPs showed less inhibition of growth as biofilm development continued. Oral Activity of an Allometrically Scaled AMP in Steers We successfully measured plasma levels of a model AMP, produced by our platform technology, in steers following oral administration. These data suggest that the evidence for small molecule transport systems in the rumen of calves and sheep (43), may account for the oral activity of our peptide drug. The AMP we used was our cyclized melanocortin antagonist; TCMCB07. We allometrically scaled the dose used in dogs to produce an increase in body weight. Allometric scaling involves the use of differences in body surface area to predict appropriate drug doses between species (29). Different drug classes show better or worse adherence to allometric scaling, depending on species-specific metabolism. Peptides are particularly good for this type of scaling; as endogenous substances in all mammals, there are similar metabolic pathways for metabolism and excretion. We previously found excellent allometric scaling for TCMCB07 (a cationic-aromatic peptide with melanocortin receptor antagonist activity) between rats and dogs in terms of plasma levels and effects on enhancing weight gain (see Fig. 4, below). Fig 4 (left) shows the effect of TCMCB07 on daily body weight (BW) in a canine 28 day safety study. Dogs were on a fixed food intake. Arrows on X axis denote when drug treatment was begun and ended. Red bars are when safety data was collected. Animals progressively gained weight on drug (becoming statistically significant by day 10, with a 13.5% increase in BW by day 24), and rapidly lost weight when drug treatment was ended (returning to baseline by day 36). This effect is analogous to (allometrically scales from) a TCMCB07-induced increase in BW of normal rats. The potential implications of finding any significant TCMCB07 oral activity in a ruminant is related to the way the oral activity of this peptide was discovered. Melanocortin antagonist effects on body weight require access to central nervous system melanocortin 4 receptors that are located behind the blood-brain-barrier [BBB (44, 45)]. We used our platform technology to develop a series of peripherally active melanocortin receptor antagonist peptides; i.e. peptides that would be transported across the BBB. The most potent of these peptides showed oral activity, suggesting a similar mechanism of transport. We've now confirmed the general aspects of our drug's oral activity and ability to enhance body weight gain in both rats and dogs, and have successfully applied the platform to produce orally active therapeutic peptides outside the cationic-aromatic class. Thus, the presence of TCMCB07 oral activity in steers suggests that AMPs produced by the platform will cross the BBB and potentially (depending on sequence) be able to interact with melanocortin receptors to control body weight.
Publications
|