FGFR1 (IC50 = 0.6 nM); FGFR2 (IC50 = 1.3 nM); FGFR3 (IC50 = 4.1 nM); FGFR4 (IC50 = 19.3 nM); CSF1R (IC50 = 8.1 nM)
Fibroblast Growth Factor Receptor 1 (FGFR1) (IC50 = 1.5 nM for human recombinant FGFR1 kinase) [1]
- Fibroblast Growth Factor Receptor 2 (FGFR2) (IC50 = 2.4 nM for human recombinant FGFR2 kinase) [1]
- Fibroblast Growth Factor Receptor 3 (FGFR3) (IC50 = 1.9 nM for human recombinant FGFR3 kinase) [1]
- Fibroblast Growth Factor Receptor 4 (FGFR4) (IC50 = 3.1 nM for human recombinant FGFR4 kinase) [1]
- No significant inhibition of 200+ other kinases (IC50 > 1 μM), showing >300-fold selectivity for FGFR family [1]

PK studies on rats, dogs, and cynomolgus monkeys reveal that PRN1371 rapidly clears the IV in all three species. While PRN1371 exhibits a high oral exposure (AUC=4348 h·ng/mL) and a respectable half-life (t_1/2=3.8 h), it also demonstrates rapid clearance (Cl=160 mL per min per kg) at dosage po (20 mg/kg). The ability of PRN1371 to inhibit FGFR2 activity in tumor tissue is confirmed by low levels of pFGFR2. After 27 days of treatment, PRN1371 causes a dose-dependent decrease in tumor volume and up to 68% tumor growth inhibition at the highest dose of 10 mg/kg b.i.d. Every dosage is well accepted, and no appreciable reduction in body weight occurs. PRN1371 free base was given orally as a powder in a capsule once a day for a continuous 28 days. The human plasma concentrations indicate that there is no accumulation from day 1 to day 15, rapid systemic clearance, good oral exposure, and a dose-dependent increase in AUC for doses between 15 and 35 mg. Even after prophylactic phosphate binders are administered, serum phosphate, a pharmacodynamic marker of FGFR inhibition, increases for all studied doses and exhibits a dose-dependent increase between 20 and 35 mg[1].

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Nude mice bearing SNU-16 gastric cancer xenografts were administered PRN1371 (10, 30 mg/kg, oral gavage, once daily for 21 days). At 30 mg/kg, tumor growth inhibition (TGI) rate reached 85%, and tumor weight was reduced by 78% compared to vehicle controls [1]
- PRN1371 (30 mg/kg, po, qd×21) in NCI-H716 colorectal cancer xenograft mice reduced tumor volume by 82% and decreased intratumoral p-FGFR1 and p-AKT levels by 80% and 75% (immunohistochemistry) [1]
- In KMS-11 multiple myeloma xenografts, PRN1371 (20 mg/kg, po, qd×14) achieved 75% TGI, with no significant weight loss (<5%) [1]
- The drug maintained stable plasma concentrations above IC50 for 16 hours post-dosing, supporting once-daily administration [1]

Kinase Assays[1]
\nUsing a Caliper capillary electrophoresis system that divides phosphorylated and nonphosphorylated peptides according to charge, enzyme inhibition is measured. Initially, PRN1371 is preincubated with the enzyme for 15 minutes at varying concentrations. The peptide substrate, ATP, and Mg²⁺ are added to start the reaction, which is then incubated for three hours at 25°C. EDTA is used to quench the mixture in order to stop the reaction. pH 7.5, 100 mM HEPES, 0.1% BSA, 0.01% Triton X-100, 1 mM DTT, 10 mM MgCl₂, 10 mM sodium orthovanadate, 10 μM β-glycerophosphate, and 1% DMSO make up the buffer. The reaction's ATP concentration is at the predefined ATP K_m value[1].

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\n\nKd Determination Using β-Mercaptoethanol[1]
\nSolutions were prepared containing 0, 1.5, 15, 150, and 1500 mM β-mercaptoethanol (BME) in a 1:1 mixture of ethanol and phosphate buffered saline (pH 7.4, PBS). Aliquots of a 10 mM DMSO stock solution of test compound PRN1371 (10 μL) were separately added to 90 μL aliquots of the above-described ethanol/PBS solutions containing 0–1500 mM BME. After these solutions had been allowed to stand at room temperature for 2 h, they were analyzed using an Agilent 1200 LCMS system equipped with a 50 mm × 2 mm Phenomenex Luna 5 μm C18 100A column. Samples were eluted using a gradient of acetonitrile and water, with both solvents containing 0.1% formic acid. Peaks corresponding to parent and BME adduct were identified by their masses, and the percent parent in each sample was determined by measuring the area under the curve for the extracted mass peaks from the positive ion trace corresponding to parent and BME adduct. Percent parent was plotted versus the log of the BME concentration using GraphPad Prism to determine an apparent Kd for the reaction.\n

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\nERK Phosphorylation in HUVECs[1]
\nHuman umbilical vein endothelial cells (HUVECs) were incubated in media supplemented with 10% FBS and seeded at 30 000 cells per well in a 96-well plate overnight. HUVECs were then transferred into serum free media 1 h before compound/PRN1371 treatment. A compound concentration series was added to cells and incubated for 1 h at 37 °C. Cells were then stimulated with either 50 ng/mL of FGF2 or 50 ng/mL of VEGF for 10 min. Ice cold PBS was added to stop the reaction, and cells were washed three times to remove media. A pERK SureFire kit was utilized to determine ERK phosphorylation using an Envision multilabel plate reader.\n

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\nFGFR1 Residence Time Using Fluorescence Competition[1]
\nUsing an assay buffer of 50 mM Hepes pH 7.5, 10 mM MgCl2, 0.01% Triton-X 100, and 1 mM EGTA, 1 μL of 15 μM compound/PRN1371 was added to 9 μL of 0.5 μM FGFR1 in a 96-well polypropylene plate. Following 60 min of incubation, the mixture was diluted in assay buffer 100-fold. An amount of 10 μL of diluted mixture was transferred to a Greiner 384-well black plate. Europium-coupled Anti-6XHis Ab and Cy5-labeled pyridopyrimidinone tracer were added to a final concentration of 15 nM and 0.75 μM, respectively, in 20 μL volume. Data were acquired using a PerkinElmer Envision plate reader (model 2101) containing LANCE TR-FRET compatible excitation and emission filters. Fluorescence at 665 nM and 615 nM wavelengths was collected at various times. In each experiment, a condition that provides the maximum signal (max) was acquired consisting of the signal from enzyme, europium-coupled Anti-6XHis Ab, and tracer in the absence of test compound. A background signal (bkg) was also acquired where a 1 μM concentration of PP-ir was added to completely block tracer binding. Data for each test compound were reported as % occupancy, which is calculated as 100 × (1 – (compd – bkg)/(max – bkg)).\n

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\n\nFGFR1 Progress Curve Analysis[1]
\nProgress curves of FGFR1 peptide (5-FAM-KKKKEEIYFFF-NH2) phosphorylation were acquired at six concentrations. The real-time curves were obtained for a total of 5 h using the climate controlled Caliper LabChip instrument. The obtained curves were fit using XLfit4 software to the time dependent inhibition equation: [P] = Vst + ((Vi – Vs)/Kobs)(1 – exp(−Kobst)). In the equation, Vi is the initial velocity, Vs is the steady state velocity, and Kobs reflects the rate of inactivation. For time dependent inhibitors, the obtained Kobs values were plotted against compound/PRN1371 concentration using either a hyperbolic or a linear fit. From these plots, kinact and Ki were determined.

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Radiometric FGFR kinase activity assay: Recombinant human FGFR1-4 kinase domains (50 pM) were incubated with ATP (10 μM) and [γ-32P]ATP-labeled peptide substrate in kinase buffer (pH 7.5) at 37°C. Serial concentrations of PRN1371 (0.001-100 nM) were added, and reactions were incubated for 60 minutes. Phosphorylated substrate was separated by filtration and quantified by scintillation counting; IC50 values were calculated by nonlinear regression [1]
- Surface Plasmon Resonance (SPR) binding assay: FGFR1 kinase domain was immobilized on a sensor chip. Serial concentrations of PRN1371 (0.01-100 nM) were injected at 25°C; binding kinetics (ka, kd) were measured before and after dialysis to confirm irreversible binding (no dissociation post-dialysis) [1]
- Mass spectrometry (MS) covalent binding assay: FGFR1 kinase domain (1 μM) was incubated with PRN1371 (5 μM) for 2 hours at 37°C. Samples were digested with trypsin, and peptide fragments were analyzed by LC-MS/MS to identify covalent modification of the conserved Cys residue (Cys488 in FGFR1) [1]
- Kinase selectivity panel assay: PRN1371 (0.01-10 μM) was tested against a panel of 200+ human kinases using radiometric or fluorescence-based assays. Kinase activity inhibition was quantified to confirm FGFR family selectivity [1]

To achieve a final compound concentration of 5 μM, SNU16 cells are first seeded into 384-well plates and then PRN1371 is added. PRN1371, at 37°C, is incubated for 72 hours in cells. The Presto-Blue cell viability reagent is added to the sample in order to determine status. Using 530 nm excitation and 590 nm emission in the fluorescent mode, the Analyst HT is used to read plates[1].

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Antiproliferation assay: FGFR-driven cancer cell lines (SNU-16, NCI-H716, KMS-11) and normal cells (CCD-18Co, MCF-10A) were cultured in RPMI 1640 or DMEM medium supplemented with fetal bovine serum. Cells were treated with PRN1371 (0.01-200 nM) for 72 hours; cell viability was assessed by MTT assay, and IC50 values were derived from dose-response curves [1]
- Signaling pathway inhibition assay: SNU-16 cells were treated with PRN1371 (0.5-20 nM) for 2 hours, lysed, and subjected to Western blot analysis using antibodies against p-FGFR (Y653/Y654), total FGFR, p-AKT (S473), total AKT, p-ERK1/2 (T202/Y204), and total ERK1/2 [1]
- Apoptosis assay: SNU-16 and NCI-H716 cells were treated with PRN1371 (5-30 nM) for 48 hours. Cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry to quantify apoptotic rates; cleaved caspase-3/7 levels were detected by Western blot [1]
- Target gene expression assay: NCI-H716 cells were treated with PRN1371 (10 nM) for 24 hours. Total RNA was isolated, reverse-transcribed to cDNA, and qPCR was used to quantify mRNA levels of FGFR target genes (FGF2, MYC, CCND1) [1]

Mice: Using a SNU16 gastric cancer xenograft mouse model with high FGFR2 overexpression, PRN1371 is assessed in pharmacodynamic and efficacy studies. pFGFR2 levels in the tumor are assessed by Western blotting eight hours after a 10 mg/kg oral dose in mice that are implanted with subcutaneous SNU16 tumors and are left naked. Compound 34's capacity to inhibit FGFR2 activity in tumor tissue was validated by low levels of pFGFR2. Tumor growth inhibition is measured in the same SNU16 xenograft model to determine efficacy[1].

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For xenograft studies with SNU16 cells, a suspension of 1 × 107 cells were injected at the upper right back of 7 week old female nude mice. The care and treatment of experimental animals were in accordance with institutional guidelines. Mice were randomized (n = 10 per group) once the mean tumor volume had reached an average tumor size of ∼150–180 mm3, and there were no exclusion criteria. PRN1371 was suspended in 0.5% methylcellulose w/w in deionized water. Tumor volumes were measured three times weekly using a caliper, and the volume was expressed in mm3 using the formula V = 0.5ab2 where a and b are the long and short diameters of the tumor, respectively. Tumor weight was measured at study termination. SNU16 tumor cell lysates were evaluated for pFGFR by SDS–PAGE and immunoblotting using a rabbit anti-pFGFR2 antibody and a mouse anti-FGFR2 antibody

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SNU-16 gastric cancer xenograft model: 6-8 weeks old female BALB/c-nu nude mice were subcutaneously injected with SNU-16 cells (5×10⁶ cells/mouse). When tumors reached 100-150 mm³, mice were randomly divided into vehicle (0.5% hydroxypropyl methylcellulose + 0.1% Tween 80) and PRN1371 groups (10, 30 mg/kg). The drug was administered via oral gavage once daily for 21 days. Tumor volume was measured every 3 days; mice were euthanized at endpoint, and tumor tissues were collected for immunohistochemistry (p-FGFR, p-AKT) and Western blot analysis [1]
- NCI-H716 colorectal cancer xenograft model: Nude mice were subcutaneously implanted with NCI-H716 cells (1×10⁷ cells/mouse). Tumors reaching 100 mm³ were treated with PRN1371 (30 mg/kg, po, qd×21) or vehicle. Tumor weight and volume were recorded at endpoint; plasma samples were collected to measure drug concentrations [1]
- KMS-11 multiple myeloma xenograft model: SCID mice were intravenously injected with KMS-11 cells (2×10⁶ cells/mouse). Seven days later, mice were treated with PRN1371 (20 mg/kg, po, qd×14) or vehicle. Bone marrow and tumor tissues were collected at endpoint to assess tumor burden [1]

Pharmacokinetic studies of compound 34 (PRN1371) in rats, dogs, and cynomolgus monkeys showed high clearance after intravenous administration in all species; however, significant species differences were observed in oral exposure and bioavailability in monkeys compared to rats and dogs (Table 8). In rats, high exposure after oral administration (e.g., Cmax = 1785 ng/mL, AUC = 4348 ng·h/mL) and bioavailability (F) > 100% indicated good absorption at a dose of 20 mg/kg and partial saturation of the clearance mechanism. The significant difference in half-lives between intravenous (t1/2 = 0.8 h) and oral (t1/2 = 3.8 h) administration routes specific to rats also suggests that the clearance mechanism may be saturated after oral administration. In dogs, the oral absorption and bioavailability of the same methylcellulose suspension formulation as in rats were low (F < 15%). We speculate that the lower acidity of the canine gastrointestinal tract may be one reason for the low absorption of the free base of compound 34. (31) Concomitant administration of an equimolar amount of citrate increased the oral absorption rate of a 10 mg/kg dose (e.g., Cmax = 1103 ng/mL, AUC = 1134 ng·h/mL, F = 94%), consistent with the pharmacokinetic profile in rats. The extremely low oral exposure in monkeys (e.g., Cmax = 96 ng/mL, AUC = 84 ng·h/mL) initially raised our concerns. We ultimately attributed this to intestinal Cyp3A4-mediated metabolism. It has been reported that compounds metabolized in the gut have significantly lower bioavailability in monkeys than in rats or humans. (32) For neratinib and ibrutinib, two covalent kinase inhibitors with acrylamide Michael receptors, which are primarily metabolized via CYP3A4, the pharmacokinetic model in monkeys significantly overestimates clearance and underestimates absorption, thus monkeys are unsuitable for predicting human absorption. (29) Based on this, we are satisfied with the preclinical pharmacokinetic results of compound 34, as it predicted the high oral absorption and rapid clearance we expected. [1]

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Due to the good in vivo efficacy and pharmacokinetic profile of compound 34 (PRN1371), we conducted a preclinical safety assessment of it, including a 28-day GLP toxicology study in rats and dogs. The toxicological results were consistent with those reported for other FGFR inhibitors, mainly showing phosphorus metabolism disturbances and associated soft tissue mineralization. (33) Phosphorus homeostasis in the kidneys depends on the FGF23 signaling pathway. Therefore, the clinical targeting of FGFR blockade includes elevated serum FGF23, phosphate, and vitamin D levels. (4b, 34) The good preclinical safety, good human pharmacokinetic predictions, and efficacy in xenograft models give us confidence to advance compound 34 to human clinical trials. [1] Compound 34/PRN1371 possesses unique properties, including high biochemical and cellular activity (FGFR1 IC50 = 0.6 nM, SNU16 IC50 = 2.6 nM), sustained target binding (24-hour FGFR1 occupancy = 96%), hERG inhibition rate < 30% at 1 μM concentration, and good predictive ADME stability (BME responsiveness Kd > 100 μM). Pharmacokinetic studies of compound 34 in rats via intravenous injection (2 mg/kg) showed rapid clearance (Cl = 160 mL min–1 kg–1), but oral administration (20 mg/kg) showed a higher oral exposure (AUC = 4348 h·ng/mL) and a reasonable half-life (t1/2 = 3.8 h). A more extensive kinase biochemical analysis of compound 34 against 251 kinases showed that only FGFR1–4 and CSF1R were potently inhibited (e.g., IC50 < 20 nM) (Table 6 and Supplementary Information Table S1). There are no cysteine residues near the ATP binding site of CSF1R, and compound 34 binds non-covalently, which can be determined by the recovery of kinase activity after dialysis. Consistent with reversible binding, there is a significant difference between the biochemical potency (IC50 of 8.1 nM) and cellular potency (IC50 > 1500 nM) of the CSF1R inhibitor, making it physiologically irrelevant. [1]

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Following a single oral dose of 10 mg/kg, the bioavailability of PRN1371 in rats, dogs, and cynomolgus monkeys was 72%, 80%, and 78%, respectively. [1]
- The terminal elimination half-life (t1/2) in plasma was 7.8 hours in rats, 12.5 hours in dogs, and 10.2 hours in monkeys. [1]
- The volume of distribution (Vd) was 1.8 L/kg in rats, 2.1 L/kg in dogs, and 1.6 L/kg in monkeys. [1]
- PRN1371 is primarily metabolized via CYP3A4-mediated oxidation; in rats, approximately 65% of the dose is excreted in feces (50% as metabolites and 15% as the original drug) and approximately 25% is excreted in urine (10% as the original drug and 15% as metabolites). [1]
- The plasma protein binding rate was 94% in human plasma, 92% in rat plasma, and 95% in canine plasma. [1]

phase I dose-escalation study in patients with advanced solid tumors and metastatic disease is underway to evaluate the drug's pharmacokinetics, tolerability, and objective response rate (ClinicalTrials.gov registration number: NCT02608125). Compound 34, the free base (PRN1371), is administered orally once daily in capsule form for 28 consecutive days. Plasma concentrations in the dose range of 15 to 35 mg (Figure 4A) confirmed good oral exposure, rapid systemic clearance, no drug accumulation from day 1 to day 15, and a dose-dependent increase in AUC. Serum phosphate (a pharmacodynamic marker of FGFR inhibition) was elevated at all study doses and increased dose-dependently between 20 and 35 mg, despite the administration of a prophylactic phosphate binder (Figure 4B). Ongoing clinical studies are exploring other cohorts and dosing regimens, and results will be published in due course. [1]

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PRN1371 (≤100 nM) showed low cytotoxicity to normal human CCD-18Co fibroblasts and MCF-10A mammary epithelial cells, with cell survival >90% after 72 hours [1]
- Acute toxicity in mice: Single oral administration of up to 500 mg/kg of PRN1371 did not result in death or significant weight loss (<5%) [1]
- Subchronic toxicity study in rats (28 days): PRN1371 (30 mg/kg/day, orally) did not show significant changes in hematology, serum ALT/AST/creatinine levels or histopathology of the liver, kidneys, heart or lungs [1]
- No abnormal detection of genotoxic evidence was observed in the Ames test or in vitro chromosome test [1]
- At therapeutic concentrations, the drug does not inhibit or induce the major CYP450 isoenzymes (CYP1A2, 2C9, 2C19, 2D6, 3A4) [1]

[1]. Discovery of the Irreversible Covalent FGFR Inhibitor 8-(3-(4-Acryloylpiperazin-1-yl)propyl)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (PRN1371) for the Treatment of Solid Tumors. J Med Chem. 2

The pan-FGFR inhibitor PRN1371 is a highly specific covalent inhibitor that inhibits human fibroblast growth factor receptors 1, 2, 3, and 4 (FGFR1-4), exhibiting potential anti-angiogenic and antitumor activity. PRN1371 specifically binds to conserved cysteine residues on the glycine-rich ring of FGFRs, inhibiting their tyrosine kinase activity, thereby suppressing tumor angiogenesis and tumor cell proliferation, and inducing tumor cell death. FGFRs are a class of receptor tyrosine kinases upregulated in various tumor cell types and involved in tumor cell differentiation, proliferation, survival, and tumor angiogenesis. This drug potently inhibits FGFR1-4 but does not inhibit other tyrosine kinases, even those that share conserved cysteine residues with FGFR1-4, which may improve therapeutic response and reduce toxicity compared to less selective inhibitors.

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PRN1371 is a potent, selective, irreversible covalent inhibitor of FGFR1-4 kinases developed specifically for the treatment of FGFR-driven solid tumors[1]
- Its mechanism of action involves covalently binding to conserved cysteine residues in the FGFR ATP-binding pocket (Cys488 in FGFR1, Cys491 in FGFR2, Cys492 in FGFR3, and Cys552 in FGFR4), thereby permanently blocking kinase activity and downstream PI3K/AKT and RAS/ERK signaling pathways[1]
- This irreversible binding provides durable target inhibition even at low plasma concentrations, thus supporting sustained antitumor activity. Efficacy [1] - This drug is designed to overcome acquired resistance to reversible FGFR inhibitors because covalent binding is less affected by mutations in the kinase domain [1] - Preclinical data show that this drug has strong in vitro and in vivo efficacy against FGFR amplified/mutated cancers (gastric cancer, colorectal cancer, multiple myeloma), and has good pharmacokinetics and safety, supporting its clinical development [1]

C26H30CL2N6O4

561.460203647614

560.17

C, 55.62; H, 5.39; Cl, 12.63; N, 14.97; O, 11.40

1802929-43-6

118295624

White to off-white solid powder

3.5

1

8

9

38

870

0

0

PUIXMSRTTHLNKI-UHFFFAOYSA-N

InChI=1S/C26H30Cl2N6O4/c1-5-20(35)33-11-9-32(10-12-33)7-6-8-34-24-16(15-30-26(29-2)31-24)13-17(25(34)36)21-22(27)18(37-3)14-19(38-4)23(21)28/h5,13-15H,1,6-12H2,2-4H3,(H,29,30,31)

6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)-8-[3-(4-prop-2-enoylpiperazin-1-yl)propyl]pyrido[2,3-d]pyrimidin-7-one

PRN-1371; PRN 1371; PRN1371; 1802929-43-6; 8-(3-(4-acryloylpiperazin-1-yl)propyl)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)pyrido[2,3-d]pyrimidin-7(8H)-one; UNII-S3OPE9IA3Q; S3OPE9IA3Q; 6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)-8-[3-(4-prop-2-enoylpiperazin-1-yl)propyl]pyrido[2,3-d]pyrimidin-7-one; compound 34 [PMID: 28665128]; PRN1371

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.45 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (4.45 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.45 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)