US 9,717,723 B2  46  45  The following materials were used:  1) CPT-11 (Irinotecan Hydrochloride) preparation having  Irinotecan Hydrochloride 98.9% by HPLC, and moisture  7 .6%. In this study drug formulations were prepared on the  "as is" basis, without correction for the moisture content or  the Irinotecan base content.  2) Liposomal CPT-11 (Ls-CPT-11) was prepared as in  Example 11, using lipid matrix of DSPC 200 mo!. parts,  Cholesterol 133  mo!.  parts, PEG-DSPE 1 mo!.  part;  entrapped solution TEA-SOS having 0.65 M TEA, pH 6.4;  drug loaded into liposomes in 5 mM HEPES buffer, 5%  dextrose, pH 6.5, at 60°  C. for 30 min at the input drug/lipid  ratio 500 mg drug/mmol of phospholipid. Loading efficiency  was >99%. Liposome size (volume average mean–standard  deviation by QELS): 101–37 nm. Liposomes were formu› lated in the vehicle, 20 mM HEPES-Na, 135 mM NaCl; pH  6.5. Drug concentrations in the injected formulations were  as stated in the Tables below.  3) Free CPT-11 solution. Free drug stock solution was  prepared by dissolving Irinotecan Hydrochloride in 5%  aqueous dextrose at 22 mg/mL, and sterilized by 0.2-µm  filtration. This stock solution was diluted with sterile 5%  dextrose prior to injection.  4) Animals. Female Swiss Webster mice, 6-8 week old,  were from Harlan, USA.  MTD determination generally followed the protocol  adopted by the United States National Cancer Institute  Developmental  Therapeutics  Program.  The  protocol  included the following three steps:  Step 1 ): Range-seeking step with the dose escalation  factor of 1.8. The groups of two animals were injected into  the tail vein with increasing doses of the free or liposomal  To obtain additional statistics for validation step, the body  weight dynamics of surviving animals was followed for up  to 11 days post injection. The dose of more than 324 mg/kg  of the liposomal Irinotecan was impossible to administer  because of the concentration and injection volume limita› tions. The results are presented in Table 10.  Step 3): Validation step. The group of5 animals is injected  i.v. (tail vein) with free or liposomal Irinotecan at tentative  MTD determined at Step 2. The animals are followed for 7  days, the animal body weight is recorded twice weekly and  compared with the pre-injection weight. General health of  the animals is observed (alertness, grooming, feeding,  excreta, skin, fur, and mucous membrane conditions, ambu› lation, breathing, posture).  If  during the observation period  there is no mortality, progressive morbidity, or weight loss  in excess of 15% of the pre-injection body weight, the dose  is considered to be validated as acute single injection MTD.  If  any of these effects occur, the experiment is repeated at the  next lower dose by a factor 1.15.  Step 2): Range-seeking step with the dose escalation  factor of 1.15. The groups of two animals were injected into  the tail vein with increasing doses of the free or liposomal  Irinotecan, beginning with the dose recorded at Step 1, and  continuing with the dose escalation factor of 1.15, until  acute mortality or terminal morbidity (within > 1 day post  injection) is observed in any of the animals. The dose one  step below the mortality/terminal morbidity dose is recorded  at tentative MTD.  Irinotecan, beginning with the dose of 60 mg/kg, and  continuing with the dose escalation factor of 1.8, until acute  mortality or terminal morbidity (within > 1 day post injec› tion) is observed in any of the animals. The dose one step  below the mortality/terminal morbidity dose is recorded.  TABLE 10  MTD seeking study of CPT-11 formulations in mice.  RESULTS  Step 1. increase dose by a factor of 1.8  Animal body weigbt, at day post injection:  inj. Dose  drug cone.  Inj. volume  mouse  0  1  2  4  5  6  7  11  drug  (mg/kg)  (mg/ml)  (µl)  #  (g)  (g)  (g)  (g)  (g)  (g)  (g)  (g)  Ls-CPTll  60  8  150  19.2  18.0  nd  20.3  20.6  20.6  20.0  19.7  2  19.7  19.3  nd  20.6  20.4  19.6  19.7  20.7  100  12  165  19.5  18.6  nd  19.6  20.0  20.1  19.4  19.9  2  20.1  18.9  nd  20.2  21.5  22.2  21.8  22.5  180  22  165  19.4  18.4  nd  18.9  19.7  20.5  19.5  20.5  2  20.0  19.3  nd  19.6  20.6  21.4  21.6  21.7  324  30.6  210  21.8  21.2  21.2  nd  20.2  nd  20.2  nd  2  21.6  20.4  21.3  nd  20.8  nd  21.4  nd  free CPTll  60  8  150  20.6  20.4  nd  22.1  22.1  22.2  22.0  22.5  2  19.5  19.1  nd  20.2  20.3  20.4  20.5  21.1  100  12  165  19.3  died 1-2 min after injection  2  20.1  died 1-2 min after injection  3  19.9  died 1-2 min after injection  Step 2. increase dose by a factor of 1.15  Animal body weight, at day post injection:  inj. Dose  drug cone.  Inj. volume  mouse  0  1  2  5  7  drug  (mg/kg)  (mg/ml)  (µl)  #  (g)  (g)  (g)  (g)  (g)  free CPTll  60  8  150  3  19.9  20.0  20.9  19.9  21.3  4  19.5  18.7  19.4  18.8  18.9  70  8  175  5  20.9  20.0  20.6  19.3  20.4  6  22.3  21.8  22.4  22.4  22.8  80  8  200  7  20.6  19.9  20.1  19.9  20.9  8  20.6  20.8  21.1  20.7  21.4 