Drug Permeability - Best Practices for Biopharmaceutics Classification System (BCS)-Based Biowaivers: A workshop Summary Report.

The workshop "Drug Permeability - Best Practices for Biopharmaceutics Classification System (BCS) Based Biowaivers" was held virtually on December 6, 2021, organized by the University of Maryland Center of Excellence in Regulatory Science and Innovation (M-CERSI), and the Food and Drug Administration (FDA). The workshop focused on the industrial, academic, and regulatory experiences in generating and evaluating permeability data, with the aim to further facilitate implementation of the BCS and efficient development of high-quality drug products globally. As the first international permeability workshop since the BCS based biowaivers was finalized as the ICH M9 guideline, the workshop included lectures, panel discussions, and breakout sessions. Lecture and panel discussion topics covered case studies at IND, NDA, and ANDA stages, typical deficiencies relating to permeability assessment supporting BCS biowaiver, types of evidence that are available to demonstrate high permeability, method suitability of a permeability assay, impact of excipients, importance of global acceptance of permeability methods, opportunities to expand the use of biowaivers (e.g. non-Caco-2 cell lines, totality-of-evidence approach to demonstrate high permeability) and future of permeability testing. Breakout sessions focused on 1) in vitro and in silico intestinal permeability methods; 2) potential excipient effects on permeability and; 3) use of label and literature data to designate permeability class.

Myeloid clonogenic assays for comparison of the in vitro toxicity of alkylating agents.

A battery of clonal assays for myeloid progenitor cells (HPP-CFC, CFU-gemm, CFU-gm, CFU-g) was utilized to evaluate the myelotoxicity of a series of alkylating agents representing the spectrum of clinical times to nadir. Bone marrow aspirates from normal volunteers were incubated with mechlorethamine, busulfan, melphalan, carmustine or lomustine for 1 h and then cultured in methylcellulose with 30% serum and cytokines. There was a concentration-dependent inhibition of colony formation and often a differential toxicity to the myeloid progenitors with the alkylators tested. On a molar basis, mechlorethamine and melphalan were the most toxic of the alkylator drugs to the myeloid precursors. The most sensitive progenitor was CFU-gemm with the lowest inhibitory concentration IC(70) concentrations for mechlorethamine, melphalan, carmustine and lomustine. Generally, there was great similarity for drug effects between CFU-g and CFU-gm with overlapping inhibition curves. HPP-CFC proved to be the least sensitive of the progenitors to the toxic actions of the drugs. While there was no correlation between the time to clinical neutropenic nadir and the most sensitive progenitor in the clonal assays, the CFU-gm assay remains a suitable method for determining the myelotoxic potential of cytotoxic agents.

Myelotoxic effects of the bifunctional alkylating agent bizelesin on human, canine and murine myeloid progenitor cells.

Bizelesin is a potent synthetic derivative of the anticancer agent CC-1065 that preferentially alkylates and binds the minor grove of DNA. Preclinical animal studies have found bizelesin to be more toxic to beagle dogs than to rodents and that myelosuppression was the dose-limiting toxicity. This toxicity was dose- and time-dependent in all species. Due to the significant difference in the in vivo myelotoxicity between species, it was important to determine which one most closely resembles humans on a pharmacodynamic basis. Therefore, hematopoietic clonal assays were utilized to evaluate the effects of bizelesin on granulocyte-macrophage (CFU-gm) colony formation. Marrow cells were exposed in vitro to bizelesin (0.001-1000 nM) for 1 or 8 h and then assayed for colony formation. There was a 3-log difference in drug concentration at which 100% colony inhibition occurred (1 or 8 h) for murine CFU-gm versus human or canine CFU-gm. The IC70 value after an 8-h bizelesin exposure for human CFU-gm (0.006 +/- 0.002 nM) was 2220-times lower than for murine CFU-gm (13.32 +/- 8.31 nM). At any given concentration, an 8 h drug exposure resulted in greater colony inhibition than a 1 h exposure for all species (P < 0.05). Increasing exposure time from 1 to 8 h increased toxicity to human and canine CFU-gm much more than to murine CFU-gm. The clinically formulated drug solution was a more potent inhibitor of human colony formation than drug dissolved in DMSO. The IC70 value after a 1-h exposure was 1.7 times lower for human CFU-gm with formulated bizelesin (0.106 +/- 0.105 nM) than bulk drug in DMSO (0.184 +/- 0.044 nM). The results of these in vitro clonal assays were qualitatively consistent with those seen in whole animal studies, suggesting that bizelesin will be a potent myelosuppressive agent in the clinic. Since the dose-limiting toxicity in preclinical models is myelosuppression and the in vitro sensitivity of human and canine CFU-gm is similar, the canine maximum tolerated dose (MTD) is better than the murine MTD to determine a safe starting dose for phase I clinical trials.

In vitro and in vivo myelotoxicity of CAI to human and murine hematopoietic progenitor cells.

Carboxyamido-triazole (CAI), an agent that targets calcium-sensitive signal transduction pathways, has both antiproliferative and antimetastatic properties. The objective of this study was to evaluate the myelotoxicity of CAI to normal human and murine hematopoietic cells. In vitro toxicity of CAI was determined by inhibition of myeloid [colony-forming unit-granulocyte/macrophage (CFU-gm)] and erythroid [burst-forming unit-erythroid (BFU-e)] colony formation in clonal assays. The effects of oral CAI on CD2F1 mouse marrow and splenic cellularity, marrow progenitor content, and peripheral blood cell counts were assessed in relation to plasma CAI levels. In vitro, CAI caused a concentration-dependent inhibition of CFU-gm and BFU-e colonies following continuous drug exposure. Murine CFU-gm and BFU-e were inhibited > 90% by 10 and 15 micrograms/mL CAI, respectively. However, suppression of human CFU-gm and BFU-e did not exceed 65% at the same concentrations. In vivo, CAI reduced the number of CFU-gm and BFU-e per femur after the initial dose and through day 4. Variations in colony inhibition paralleled changes in CAI plasma concentrations. While colony inhibition increased in vitro with escalating drug concentrations, this was not observed in vivo with additional CAI doses. The low toxicity of CAI in vivo combined with the significant difference between toxicity for human and mouse progenitors in vitro suggests a relatively low adverse potential to the bone marrow for this new signal transduction inhibitory agent.

In vivo-in vitro correlation of myelotoxicity of 9-methoxypyrazoloacridine (NSC-366140, PD115934) to myeloid and erythroid hematopoietic progenitors from human, murine, and canine marrow.

BACKGROUND: 9-Methoxypyrazoloacridine (PZA) is an anticancer agent that shows selectivity of action for carcinomas over leukemias. It also has nearly equal potency against cycling and quiescent or hypoxic and normoxic target cells. Phase I trials of PZA in humans are nearing completion. PURPOSE: This study was conducted to determine (a) if PZA is directly inhibitory to hematopoietic cells and, if it is, to characterize the inhibition pharmacodynamically, (b) whether species-specific differences in direct toxicity could explain differences in myelosuppression in mice, dogs, and humans, and (c) whether in vitro data correlate with in vivo myelosuppression data. METHODS: In vitro clonogenic assays of hematopoietic progenitors of myeloid and erythroid lineages from human, canine, and murine femoral marrow were used to measure the direct toxicity of PZA. Results from these assays were compared on an area-under-the-curve (AUC) basis to clinical myelosuppression data. RESULTS: On the basis of maximum tolerated concentrations, canine hematopoietic progenitors are most susceptible to PZA, followed by human and then murine progenitors. We found no difference in susceptibility to PZA toxicity between the human progenitors of myeloid and erythroid lineages. Both concentration and duration of exposure contribute to the in vitro toxicity of PZA. In contrast to antimetabolites, the in vitro toxicity of PZA could be minimized at a given AUC by lowering drug concentration and prolonging the period of exposure. On an AUC basis, the in vitro data are consistent with limited in vivo myelosuppression data from preclinical models and correlate with neutropenia data from a phase I trial. CONCLUSIONS: PZA directly inhibits hematopoietic progenitors, an action that is responsible for the myelosuppression observed in humans. Human marrow appears able to compensate for the loss of up to 35% of its myeloid progenitors, in that peripheral neutrophil counts remain unchanged at that level of loss. Although in vivo studies show that prolonged infusion reduces myelosuppression at a given total dose in both rodent and canine models, pharmacokinetic differences make it unlikely that this approach will benefit human patients. IMPLICATIONS: The in vitro data quantitatively predict the AUCs at maximum tolerated dose in preclinical models and human patients. Thus, in vitro clonogenic assays of myelotoxic agents can provide data that make both preclinical toxicology testing and clinical trial planning and interpretation more efficient and accurate.

In vitro characterization of the myelotoxicity of cyclopentenyl cytosine.

We studied the toxicity of a new experimental anticancer drug, cyclopentenyl cytosine (CPE-C), to human and murine hematopoietic progenitor cells in vitro. Due to CPE-C's in vivo myelotoxicity, it was important to characterize its potential adverse effects on human marrow cells during preclinical development of the drug. Marrow cells were exposed to CPE-C for either 1 h prior to addition in clonal assays or continuously during their culture period. The inhibitory effects of CPE-C on myeloid (CFU-gm) and erythroid (CFU-e, BFU-e) colony formation were concentration- and time-dependent, with continuous CPE-C exposure being significantly more inhibitory than 1-h exposure. The results of both exposure experiments were combined to investigate colony inhibition as a function of overall drug exposure (concentration x time, AUC) and data analyzed by the nonlinear Emax equation. Human and murine CFU-gm had similar AUC-response curves and IAUC70 values (i.e., AUC at 70% colony inhibition) of 40.8 and 41.9 microM h, respectively. In contrast, murine CFU-e and BFU-e were more sensitive to CPE-C, having lower IAUC70 values (both, 21.1 microM h) than human CFU-e and BFU-e (107.8 and 33.0 microM h, respectively). This difference was most prominent with the late erythroid progenitor, CFU-e, in that the human cells were 5 times more resistant to inhibition by CPE-C. CPE-C was myelotoxic in vitro to human and murine marrow cells and toxicity correlated with overall drug exposure.

Myelotoxicity of rifabutin and 3'-azido-3'-deoxythymidine, alone and in combination, to human hematopoietic progenitor cells in vitro.

Mycobacterial infection is a common complication of acquired immunodeficiency syndrome (AIDS) frequently requiring antimycobacterial medication. It was of interest to determine if one such agent, rifabutin, could be tolerated by AIDS patients in conjunction with 3'-azido-3'-deoxythymidine (AZT) therapy. We evaluated the in vitro myelotoxic effects of rifabutin on human hematopoietic progenitor cells, alone and in combination with AZT (rifabutin: AZT, 1:10 ratio) over a range of concentrations in a microcapillary assay. Both rifabutin and AZT at 5 microM were moderately toxic to hematopoietic progenitors, inhibiting colony formation by 57-65% and 59-63%, respectively. The combination of rifabutin (5 microM) and AZT (50 microM) inhibited colony formation by 59-73%. Granulocyte-macrophage progenitors were less sensitive to this combination than erythroid progenitors. The combination of ribabutin and AZT did not exceed the in vitro myelotoxicity to human progenitors of AZT alone. These results suggest that rifabutin may be tolerated in AIDS patients, with no anticipated increase in myelotoxicity when given with AZT.

Comparative in vitro myelotoxicity of FCE 24517, a distamycin derivative, to human, canine and murine hematopoietic progenitor cells.

FCE 24517, a derivative of distamycin A, exhibits an unusual antitumor profile in experimental models. As part of its preclinical development, we evaluated the in vitro myelotoxicity of FCE 24517 to human, canine and murine hematopoietic cells. Marrow cells were exposed to the agent (2.7 x 10(-5) - 2.7 nM) for 4 h and then assayed in capillary (human) or Petri dish (canine, murine) clonal cultures. FCE 24517 inhibited myeloid (CFU-gm), erythroid (BFU-e, CFU-e) and megakaryocytic (CFU-meg) colony formation in a concentration-dependent manner. The progenitor cells were generally similar in their response to FCE 24517 within a species. Comparing the different progenitor cell response to FCE 24517, canine CFU-gm and CFU-e were 26- to 221-fold more sensitive to this drug's toxic effects than their human and murine counterparts. This was demonstrated by extremely low IC70 values for the canine CFU-gm (0.001 nM) and CFU-e (0.007 nM). Murine progenitors displayed 1.3- to 10.9-times higher IC70 values than human CFU-gm, BFU-e and CFU-e following 4 hr exposure to FCE 24517. The data demonstrated that a mouse model may better predict human in vitro myelotoxicity to FCE 24517 than beagle dogs.

In vitro toxicity of 3'-azido-3'-deoxythymidine, carbovir and 2',3'-didehydro-2',3'-dideoxythymidine to human and murine haematopoietic progenitor cells.

The myelotoxicities of three antiretroviral agents, 3'-azido-3'-deoxythymidine (AZT), carbovir (CBV) and 2',3'-didehydro-2',3'-dideoxythymidine (d4T), were evaluated in vitro with normal human and murine haematopoietic progenitor cells. These studies demonstrated that continuous AZT exposure was more inhibitory to human and murine colony formation than 1 h exposure, with murine and human progenitors similarly inhibited by continuous AZT exposure. These in vitro results on AZT's myelotoxicity correlate with both human and murine in vivo studies. CBV was only moderately toxic to human and murine cells following either 1 h or continuous exposure, with human and murine progenitors similarly suppressed by continuous CBV exposure. 1 h d4T exposure was less toxic to both human and murine marrow cells than continuous exposure and both species were equivalently inhibited when continuously exposed to d4T. In general, CBV was the least toxic agent to human and murine haematopoietic cells and AZT the most toxic. The study establishes CBV and d4T as less myelotoxic agents to human and murine haematopoietic progenitor cells in vitro than AZT which therefore could be considered as alternatives to AZT for the treatment of HIV infection.

Comparison of the in vitro toxicity of 2',3'-dideoxynucleosides to murine hematopoietic progenitor cells.

Four nucleoside analogues, 2',3'-dideoxyinosine (ddI), 2',3'-dideoxyadenosine (ddA), 2',3'-dideoxycytosine (ddC) and 5-fluoro-2',3'-dideoxycytosine (5-F-ddC), were evaluated for their potential in vitro myelotoxic effects on normal murine hematopoietic progenitor cells. Myeloid granulocyte-macrophage colony-forming units (CFU-gm), erythroid burst-forming units (BFU-e) and colony-forming units (CFU-e) and megakaryocytic (CFU-meg) progenitors were exposed to the agents for 1 h prior to culture in Petri dish assays or continuously throughout the entire culture period. At 10 microM, both ddA and ddI were moderately toxic (2-36% colony inhibition) to murine CFU-gm, BFU-e, CFU-e and CFU-meg following either 1 h or continuous exposure. Colony inhibition for the progenitors ranged from 2-31% at 10 microM for 1 h ddC or 5-F-ddC exposure. Continuous exposure to ddC was highly myelotoxic to murine hematopoietic progenitors with 100 microM suppressing colony formation 82-89%. At the same concentration and exposure time, 5-F-ddC inhibited colony formation 56-67%. Our results demonstrate that 1 h and continuous exposures to ddA and ddI were similarly myelotoxic to murine hematopoietic cells regardless of exposure time. In contrast, continuous ddC or 5-F-ddC exposure was more toxic to murine progenitors than 1 h exposure to these agents.

Comparative toxicity of fostriecin, hepsulfam and pyrazine diazohydroxide to human and murine hematopoietic progenitor cells in vitro.

The in vitro myelotoxic potentials of three investigational antitumor agents, Fostriecin, Hepsulfam and pyrazine diazohydroxide (PZDH), were evaluated utilizing clonogenic assays. Human and murine marrow cells were exposed to each drug for 1 hr prior to culture in microcapillary (human) or Petri dish (murine) assays. Fostriecin (0.22-220 microM), Hepsulfam (0.34-340 microM) and PZDH (0.68-680 microM) inhibited myeloid (CFU-gm), erythroid (BFU-e, CFU-e) and megakaryocytic (CFU-meg) colony formation in a concentration-dependent manner. CFU-e from both species were more sensitive to Fostriecin than the other progenitors and murine cells more sensitive overall to Fostriecin than their human counterparts. Murine CFU-e were also more sensitive to Hepsulfam than human CFU-e, with CFU-gm and BFU-e being similarly affected in both species. Human BFU-e were greatly inhibited by PZDH, whereas murine BFU-e were relatively resistant to its toxic effects. Fostriecin was the most toxic of the three antitumor agents, with PZDH the least toxic.

Utility of human bone marrow obtained incidental to orthopedic surgery for hematopoietic clonal assays.

We describe the establishment of a convenient method of acquiring human bone marrow cells for use in microcapillary clonal assays. Femoral epiphyseal and diaphyseal bone fragments and femoral canal reamings were collected incidental to total hip replacement surgery and cultured for human granulocyte-macrophage colony-forming units, erythroid burst-forming units and erythroid colony-forming units. This readily available source of normal human marrow provides an abundant quantity of hematopoietic progenitors of documented normalcy.

In vitro myelotoxicity of 2',3'-dideoxynucleosides on human hematopoietic progenitor cells.

Three nucleoside analogues, 2',3'-dideoxyadenosine (ddA), 2',3'-dideoxyinosine (ddI), and 2',3'-dideoxycytosine (ddC), were evaluated for their potential myelotoxic effects to normal human hematopoietic progenitor cells. The myeloid (granulocyte-monocyte colony-forming units, CFU-gm) and erythroid (erythroid burst-forming units, BFU-e: and erythroid colony-forming units, CFU-e) committed progenitor cells were exposed to the agents for a 1-h period prior to culture in a microcapillary assay or continuously exposed during the entire culture period. Both ddA and ddI (100 microM) were mildly toxic (less than 50% colony inhibition) to human CFU-gm, BFU-e, and CFU-e following either 1-h or continuous exposures. Marrow progenitor sensitivities to ddA and ddI were indistinguishable. Colony inhibition ranged from 47% to 67% for 1-h ddC exposure (100 microM), values that were comparable to ddA and ddI. Continuous exposure to ddC was highly myelotoxic to human hematopoietic progenitors, with concentrations of 10 and 100 microM suppressing colony formation by 79%-92% and 93%-97%, respectively. These results demonstrate that 1-h and continuous exposures to ddA and ddI were similarly myelotoxic to human hematopoietic cells, whereas a 1-h exposure to ddC was equivalent to ddA and ddI, yet continuous ddC exposure was extremely toxic to marrow cell progenitors.

Effects of L-phenylalanine mustard and L-buthionine sulfoximine on murine and human hematopoietic progenitor cells in vitro.

The effects of L-buthionine sulfoximine (L-BSO) and L-phenylalanine mustard (L-PAM), alone and in combination, on human and murine marrow were explored using in vitro clonogenic assays to establish whether enhanced myelotoxicity might limit the clinical utility of this potent chemotherapeutic combination. One-h exposure to L-PAM produced significant concentration-dependent colony inhibition, with 70% inhibitory concentration (IC70) values ranging from 4.5 to 7.2 microM for all hematopoietic progenitors assayed. The combination of L-PAM plus 4500 microM L-BSO for 1 h did not effectively alter the IC70 values derived for L-PAM alone. In studies where marrow cells were pretreated with L-BSO for 4 h and then L-PAM for 1 additional h, the IC70 values were decreased in both murine and human marrow progenitors compared to the L-PAM control, suggesting modest potentiation of myelotoxicity. The potentiation is not so significant as to preclude human studies with this combination. One- to 5-h exposure of marrow cells from both species to 4500 microM L-BSO was only mildly myelotoxic, producing colony reductions of 22-49%. However, continuous exposure to L-BSO produced concentration-dependent colony inhibition, with IC70 values of 70, 84, and 43 microM for murine colony-forming units-granulocyte/macrophage, blast-forming units-erythroid, and colony-forming unit-erythroid, respectively.

Microcapillary clonogenic assays for human marrow hematopoietic progenitor cells.

The capillary clonogenic cell assay was developed and adapted to culture myeloid and erythroid colonies from human bone marrow cells. The plating efficiencies for femoral bone marrow granulocyte-macrophage progenitors (CFU-gm), erythroid colony-forming units (CFU-e) and erythroid burst-forming units (BFU-e) were 0.143%, 0.229% and 0.141%, respectively. Standard bone marrow progenitor Petri dish assays require a total culture volume of 1 ml per dish, and as such are not suitable for the small numbers of cells often obtained from human bone marrow samples. The microcapillary assay as developed and standardized in our laboratory has the unique advantage of being able to utilize small numbers of cells. This technique is suitable for evaluating the myelotoxicity of investigational new anti-cancer and anti-HIV agents and for further investigation of the mechanisms underlying chemotherapy-induced bone marrow toxicity.