Susan Pugh

Formulation and Antitumor Activity Evaluation of Nanocrystalline Suspensions of Poorly Soluble Anticancer Drugs

AbstractPurpose. Determine if wet milling technology could be used to formulate water insoluble antitumor agents as stabilized nanocrystalline drug suspensions that retain biological effectiveness following intravenous injection. Methods. The versatility of the approach is demonstrated by evaluation of four poorly water soluble chemotherapeutic agents that exhibit diverse chemistries and mechanisms of action. The compounds selected were: piposulfan (alkylating agent), etoposide (topoisomerase II inhibitor), camptothecin (topoisomerase I inhibitor) and paclitaxel (antimitotic agent). The agents were wet milled as a 2% w/v solids suspension containing 1 % w/v surfactant stabilizer using a low energy ball mill. The size , physical stability and efficacy of the nanocrystalline suspensions were evaluated. Results. The data show the feasibility of formulating poorly water soluble anticancer agents as physically stable aqueous nanocrystalline suspensions. The suspensions are physically stable and efficacious following intravenous injection. Conclusions. Wet milling technology is a feasible approach for formulating poorly water soluble chemotherapeutic agents that may offer a number of advantages over a more classical approach.

In Vivo Methods for Screening and Preclinical Testing

The classic question in the field of drug discovery is: Which tumor model is a satis-factory predictor for cancer in humans? The classic answer is: None of them!

Preclinical antitumor efficacy of analogs of XK469: sodium-(2-[4-(7-chloro-2-quinoxalinyloxy)phenoxy]propionate

A series of quinoxaline analogs of the herbicide Assure® was found to have selective cytotoxicity for solid tumors of mice in a disk-diffusion-soft-agar-colony-formation-assay compared to L1210 leukemia. Four agents without selective cytotoxicity and 14 agents with selective cytotoxicity were evaluated in vivo for activity against a solid tumor. The four agents without selective cytotoxicity in the disk-assay were inactive in vivo (T/C > 42%). Thirteen of the fourteen agents with selectivity in the disk-assay were active in vivo (T/C < 42%). Five of the agents had curative activity. These five agents had a halogen (F, Cl, Br) in the 7-position (whereas Assure® had a Cl in the 6 position). All agents with curative activity were either a carboxylic acid, or a derivative thereof, whereas Assure® is the ethyl ester of the carboxylic acid. All other structural features were identical between Assure® and the curative agents. Assure® had no selective cytotoxicity for solid tumors in the disk-assay, and was devoid of antitumor activity. The analog XK469 is in clinical development.

Cryptophycins-309, 249 and other cryptophycin analogs: preclinical efficacy studies with mouse and human tumors.

SummaryCryptophycins-1 and 52 (epoxides) were discovered to have in-vitro and in-vivo antitumor activity in the early 1990s. The chlorohydrins of these, Cryptophycins-8 and 55 (also discovered in the early 1990s) were markedly more active, but could not be formulated as stable solutions. With no method to adequately stabilize the chlorohydrins at the time, Cryptophycin-52 (LY 355073) entered clinical trials, producing only marginal antitumor activity. Since that time, glycinate esters of the hydroxyl group of the chlorohydrins have been synthesized and found to provide stability. Three of the most active were compared herein. Cryptophycin-309 (C-309) is a glycinate ester of the chlorohydrin Cryptophycin-296. The glycinate derivative provided both chemical stability and improved aqueous solubility. After the examination of 81 different Cryptophycin analogs in tumor bearing animals, C-309 has emerged as superior to all others. The following %T/C and Log Kill (LK) values were obtained from a single course of IV treatment (Q2d × 5) against early staged SC transplantable tumors of mouse and human origin: Mam 17/Adr [a pgp (+) MDR tumor]: 0%T/C, 3.2 LK; Mam 16/C/Adr [a pgp (−) MDR tumor]: 0%T/C, 3.3 LK; Mam 16/C: 0%T/C, 3.8 LK; Colon 26: 0%T/C, 2.2 LK; Colon 51: 0%T/C, 2.4 LK; Pancreatic Ductal Adenocarcinoma 02 (Panc 02): 0%T/C, 2.4 LK; Human Colon HCT15 [a pgp (+) MDR tumor]: 0%T/C, 3.3 LK; Human Colon HCT116: 0%T/C, 4.1 LK. One additional analog, Cryptophycin-249 (C-249, the glycinate of Cryptophycin-8), also emerged with efficacy rivaling or superior to C-309. However, there was sufficient material for only a single C-249 trial in which a 4.0 LK was obtained against the multidrug resistant breast adenocarcinoma Mam-16/C/Adr. C-309 and C-249 are being considered as second-generation clinical candidates.

Treatment of human prostate tumors PC-3 and TSU-PR1 with standard and investigational agents in SCID mice

Both the PC-3 and the TSU-PR1 prostate tumor models were found to be satisfactory for chemotherapeutic investigations in ICR-SCID mice. The 30 to 60 mg fragments implanted took in all mice (as judged by 100% takes in the controls of all experiments as well as the passage mice). The tumor volume doubling time was 4.0 days for PC-3 and 2.5 days for TSU-Pr1. Nine agents were evaluated IV against early stage subcutaneous PC-3 tumors, with Nano-piposulfan being the only agent highly active (4.9 log kill). Three other agents were moderately active: Taxol (1.5 log kill), Cryptophycin-8 (1.6 log kill), Vinblastine (1.0 log kill). Five agents were inactive: VP-16, Adriamycin, CisDDPt, 5-FUra, and Cyclophosphamide. Ten agents were evaluated IV against early stage subcutaneous TSU-Pr1 tumors. Three agent were highly active, producing > 6 log kill and cures: Taxol (5/5 cures), Cryptophycin-8 (5/5 cures), Vinblastine (2/4 cures). Two other agents were moderately active: Nano-piposulfan (1.2 log kill), and Cyclophosphamide (1.1 log kill). Five agents were inactive: VP-16, Adriamycin, CisDDPt, 5-FUra, and BCNU. In part, activity was determined by the ability of the SCID mice to tolerate meaningful dosages of the agents. Agents producing granulocyte toxicity (e.g., Adriamycin) were poorly tolerated and appeared less active than expected. Vinblastine, producing little or no granulocyte toxicity was very well tolerated and appeared to be more active than expected.

Discovery of cryptophycin-1 and BCN-183577 : Examples of strategies and problems in the detection of antitumor activity in mice

Historically, many new anticancer agents were first detected in a prescreen; usually consisting of a molecular/biochemical target or a cellular cytotoxicity assay. The agent then progressed to in vivo evaluation against transplanted human or mouse tumors. If the investigator had a large drug supply and ample resources, multiple tests were possible, with variations in tumor models, tumor and drug routes, dose-decrements, dose-schedules, number of groups, etc. However, in most large programs involving several hundred in vivo tests yearly, resource limitations and drug supply limitations have usually dictated a single trial. Under such restrictive conditions, we have implemented a flexible in vivo testing protocol. With this strategy, the tumor model is dictated by in vitro cellular sensitivity; drug route by water solubility (with water soluble agents injected intravenously); dosage decrement by drug supply, dose-schedule by toxicities encountered, etc. In this flexible design, many treatment parameters can be changed during the course of treatment (e.g., dose and schedule). The discovery of two active agents are presented (Cryptophycin-1, and Thioxanthone BCN 183577). Both were discovered by the intravenous route of administration. Both would have been missed if they were tested intraperitoneally, the usual drug route used in discovery protocols. It is also likely that they would have been missed with an easy to execute fixed protocol design, even if injected IV.

Discovery of Solid Tumor Active Agents Using a Soft-Agar-Colony-Formation Disk-Diffusion-Assay

The history of antitumor drug discovery has essentially been the use of two lymphocytic leukemias of mice as selection funnels through which all agents needed to pass in order to advance toward clinical development (L1210 prior to 1975 and P388 after 1975). It is thus not surprising that agents in the clinic are highly active against these tumor systems. However, none of the agents discovered by these leukemias are tumor specific (i.e., active against all tumors), and none of the agents are broadly active against solid tumors of either rodents or humans (1, 2, 3). An example contrasting the responsiveness of transplantable solid tumors of mice and the two leukemias is shown in Table-1. The lack of responsiveness of these solid tumors of mice is not unlike those seen in human lung, pancreatic, colon, and prostate tumors. The point to emphasize is that the lack of solid tumor activity of available antitumor agents is not species related. The fault does not lie with the omission of human tumors in the initial selection process, but rather with the omission of solid tumors.

Tumor Models and the Discovery and Secondary Evaluation of Solid Tumor Active Agents

AbstractEach independently arising tumor is a separate and unique biologic entity with its own unique histologic appearance, biologic behavior, and drug response profile. Thus, in drug discovery, no single tumor has been a perfect predictor for any other tumor. For this reason, new agents are evaluated in a variety of tumor models which is known as breadth of activity testing. In recent years, human tumors implanted in athymic nude mice and SCID mice have also become available for breadth of activity testing. In studies carried out in these laboratories, it was found that 10 human tumors metastasized in the SCID mice, but failed to metastasize in nude mice. In addition, tumor growth and tumor takes were superior in the SCID mice. The strengths and weaknesses of xenograft model systems are discussed. For example, most human tumor xenograft models are excessively sensitive to alkylating agents as well as to a new class of DNA binders (XE840 and XP315). Using human tumor models that are the least sensitive t...

Preclinical efficacy of thioxanthone SR271425 against transplanted solid tumors of mouse and human origin

A highly active and broadly active thioxanthone has been identified: N-[[1-[[2-(Diethylamino)ethyl]amino]-7-methoxy-9-oxo-9H-thioxanthen-4-yl] methylformamide (SR271425, BCN326862, WIN71425). In preclinical testing against a variety of subcutaneously growing solid tumors, the following %T/C and Log10 tumor cell kill (LK) values were obtained: Panc-03 T/C = 0, 5/5 cures; Colon-38 (adv. stage) T/C = 0, 3/5 cures, 4.9 LK; Mam-16/C T/C = 0, 3.5 LK; Mam-17/0 T/C = 0, 2.8 LK; Colon-26 T/C = 0, 1/5 cures, 3.2 LK; Colon-51 T/C= 0, 2.7 LK; Panc-02 T/C = 0, 3.1 LK; B16 Melanoma T/C = 13%, 4.0 LK; Squamous Lung-LC12 (adv. stage) T/C = 14%, 4.9 LK; BG-1 human ovarian T/C = 16%, 1.3 LK; WSU-Br1 human breast T/C = 25%, 0.8 LK. The agent was modestly active against doxorubicin (Adr)-resistant solid tumors: Mam-17/Adr T/C =23%, 0.8 LK; and Mam-16/C/Adr T/C = 25%, 1.0 LK, but retained substantial activity against a taxol-resistant tumor: Mam-16/C/taxol T/C = 3%, 2.4 LK. SR271425 was highly active against IV implanted leukemias, L1210 6.3 LK and AML1498 5.3 LK. The agent was equally active both by the IV and oral routes of administration, although requiring approximately 30% higher dose by the oral route. Based on its preclinical antitumor profile, it may be appropriate to evaluate SR271425 in clinical trials.

Preclinical efficacy evaluations of XK-469: Dose schedule, route and cross-resistance behavior in tumor bearing mice

XK-469 is advancing to Phase I clinicaltrials. Preclinical studies were carriedout to assist in clinical applications.Dose-schedule route testing: Singledose IV treatment with XK-469 producedlethality (LD20 to LD 100) above 142 mg/kg.Optimum treatment required total dosages of350 to 600 mg/kg. Furthermore, highindividual IV dosages (100 to 142 mg/kg)were poorly tolerated, producingsubstantial weight loss (8 to 18% of bodyweight), poor appearance, and slow recovery(8 to 12 days). A 1-hour infusion ofdosages more than 140 mg/kg, or BIDinjections 6 hrs apart, did not reducelethality. However, lower individualdosages of 40 to 50 mg/kg/injection IV werewell tolerated and could be given daily toreach an optimum total dose with minimaltoxicities. Likewise, 75 mg/kg/injection IVcould be used every other day to reachoptimal treatment. The necropsy profiles ofdeaths from toxic dosages were essentiallyidentical regardless of schedule (deaths 4to 7 days post treatment). The profileswere: paralytic ileus or gastroparesis; GIepithelial damage; and marrow toxicity.Interestingly, the key lethal events wererapidly reversible and simple to overcomewith lower dosages given daily or everyother day. Based on these results, the highdose, Q21day schedule should be avoided inclinical applications. Instead, a splitdose regimen is recommended (e.g., daily,every other day, or twice weekly). XK-469was also well tolerated by the oral route,requiring 35% higher dosages PO to reachthe same efficacy and toxicity as producedIV. Cross-resistance studies: XK-469resistance was produced by optimumtreatments of IV implanted L1210 leukemiaover seven passage generations. Thisleukemia subline (L1210/XK469) had reducedsensitivity to VP-16 (with a 4.0 log killin IV implanted L1210/XK469 compared to an8.0 log kill against IV implanted L1210/0). It also had a reduction in the sensitivityto 5-FU (with a 2.0 log kill in theimplanted L1210/XK469 compared to a 4.0 logkill against IV implanted L1210/0). Otheragents were approximately as active againstthe resistant tumor, including: Ara-C,Gemzar, Cytoxan, BCNU, DTIC, and CisDDPT. No case of collateral sensitivity wasobserved; i.e., no agent was markedly moreactive against the resistant sublineL1210/XK-469 than against the parent tumorin mice.