The FDA has published recommendations for sponsors who wish to request a waiver of in vivo bioavailability (BA) or bioequivalence (BE) studies for immediate release (IR) solid oral dosage forms based on the Biopharmaceutics Classification System (BCS). Biowaivers can be requested for IR formulations in which the active ingredient is shown to be a BCS class I drug: that is, a drug showing high permeability and high solubility over a pH range of 1-7.5. For permeability determinations, a variety of experimental methods can be used, such as the rat in situ single pass perfusion or Caco-2 cell culture models, once the suitability of the particular method is established. Following the recommended procedure for assessing the suitability of permeability determinations, we determined the permeability of 20 test drugs using the in situ single pass perfusion model in rats. The test compounds were coperfused through jejunal intestinal segments with an internal permeability reference standard (metoprolol) over a 90 min time period. Sample analysis was performed by HPLC, and the ratio of the effective permeability, Peff (cm/s), of test compound to that of metoprolol was determined. To address the question of test drug permeabilities that approach that of the internal standard, we propose that a statistical analysis such as the "0.8-1.25 rule" used for in vivo or in vitro bioequivalence studies provide guidance for permeability classification using the in situ single pass perfusion model. We developed a method using the 90% confidence interval of the permeability ratio of the test to internal reference standard in order to differentiate between high and low permeability compounds. This analysis allowed for the proper permeability classification of all of the test compounds and suggests a robust means for assessing drug permeability classification.

Cidofovir (HPMPC, 1), a broad-spectrum antiviral agent, is currently used to treat AIDS-related human cytomegalovirus (HCMV) retinitis and has recognized therapeutic potential for orthopox virus infections, but is limited by its low oral bioavailability. Cyclic cidofovir (2) displays decreased nephrotoxicity compared to 1, while also exhibiting potent antiviral activity. Here we describe in detail the synthesis and evaluation as prodrugs of four cHPMPC dipeptide conjugates in which the free POH of 2 is esterified by the Ser side chain alcohol group of an X-L-Ser(OMe) dipeptide: 3 (X = L-Ala), 4 (X = L-Val), 5 (X = L-Leu), and 6 (X = L-Phe). Perfusion studies in the rat establish that the mesenteric permeability to 4 is more than 30-fold greater than to 1, and the bioavailability of 4 is increased 8-fold relative to 1 in an in vivo murine model. In gastrointestinal and liver homogenates, the cHPMPC prodrugs are rapidly hydrolyzed to 2. Prodrugs 3, 4, and 5 are nontoxic at 100 μM in HFF and KB cells and in cell-based plaque reduction assays had IC 50 values of 0.1-0.5 μM for HCMV and 10 μM for two orthopox viruses (vaccinia and cowpox). The enhanced transport properties of 3-6, conferred by incorporation of a toxicologically benign dipeptide moiety, and the facile cleavage of the Ser-O-P linkage suggest that these prodrugs represent a promising new approach to enhancing the bioavailability of 2.

The interplay between gastrointestinal physiology (lower CYP 3A expression in the distal ileum and the colon) and formulation design (zero-order controlled release after a predetermined lag-time) resulted in successful absorption and bioavailability improvement and represent a viable strategy to reduce the dose of CYP 3A drugs.

Cidofovir (HPMPC) is a broad-spectrum anti-viral agent whose potential, particularly in biodefense scenarios, is limited by its low oral bioavailability. Two prodrugs (3, 4) created by conjugating ethylene glycol-linked amino acids (L-Val, L-Phe) with the cyclic form of cidofovir (cHPMPC) via a P-O ester bond were synthesized and their pH dependent stability (3, 4) potential for in vivo reconversion to drug (3), and oral bioavailability (3) were evaluated. The prodrugs were stable in buffer between pH 3-5, but underwent rapid hydrolysis in liver (t ½ = 3.7 min), intestinal (t ½ = 12.5 min) and Caco-2 cell homogenates (t ½ = 20.2 min). In vivo (rat), prodrug 3 was >90% reconverted to cHPMPC. The prodrug was 4x more active than ganciclovir (IC 50 value, 0.68 μM vs 3.0 μM) in a HCMV plaque reduction assay. However, its oral bioavailability in a rat model was similar to the parent drug. The contrast between the promising activation properties and unenhanced transport of the prodrug is briefly discussed. KeywordsProdrug; cHPMPC; HPMPC; biotransformation; HCMV; anti-viral; biodefense Cidofovir (Vistide®, HPMPC, 1, Fig. 1) is a broad-spectrum anti-viral agent that is used in the treatment of cytomegalovirus (CMV) retinitis in AIDS patients. Cidofovir also has therapeutic potential in the treatment of other herpes and DNA viruses including polyoma-, papilloma-, adeno-, and poxvirus infections. 1, 2 Recently, cidofovir has become of particular interest as a potential emergency therapy for orthopox virus infections. 3 An important limitation of cidofovir and analogous nucleotide drugs in a therapeutic role is their low oral bioavailability and poor transport into cells, necessitating intravenous administration. In an emergency situation such as a smallpox outbreak, an orally available form of cidofovir would thus be highly advantageous.Our current research is focused on improving cidofovir's oral bioavailability. The phosphonic diacid group of cidofovir is ionized under physiological conditions, which substantially accounts for its low bioavailability (<5%). 4, 5 Cyclic cidofovir (cHPMPC, 2 , Fig. 1 ), undergoesCorresponding Author: Dr. Charles McKenna, Univ. of Southern Calif. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript biotransformation when exposed to cCMP phosphodiesterase to generate the parent drug, HPMPC 6, 7 . Severe nephrotoxic effects have been associated with cidofovir treatment 8 while cHPMPC has been reported to be significantly less nephrotoxic. 6 cHPMPC, also exhi...