HIV-1
Fostemsavir (BMS-663068): Prodrug of BMS-626529; the active component BMS-626529 targets HIV-1 envelope glycoprotein gp120 (binds to the CD4-binding site of gp120, EC50 for wild-type HIV-1 IIIB in TZM-bl cells = 0.035 μM; Ki for gp120-CD4 binding = 0.012 μM) [1]

BMS-663068 is a prodrug of the small-molecule inhibitor Temsavir/BMS-626529, which inhibits human immunodeficiency virus type 1 (HIV-1) infection by binding to gp120 and interfering with the attachment of virus to CD4+ T-cells.The activity of BMS-626529 is virus dependent, due to heterogeneity within gp120. In order to better understand the anti-HIV-1 spectrum of BMS-626529 against HIV-1, in vitro activities against a wide variety of laboratory strains and clinical isolates were determined. BMS-626529 had half-maximal effective concentration (EC(50)) values of <10 nM against the vast majority of viral isolates; however, susceptibility varied by >6 log(10), with half-maximal effective concentration values in the low pM range against the most susceptible viruses. The in vitro antiviral activity of BMS-626529 was generally not associated with either tropism or subtype, with few exceptions. Measurement of the binding affinity of BMS-626529 for purified gp120 suggests that a contributory factor to its inhibitory potency may be a relatively long dissociative half-life. Finally, in two-drug combination studies, BMS-626529 demonstrated additive or synergistic interactions with antiretroviral drugs of different mechanistic classes. These results suggest that BMS-626529 should be active against the majority of HIV-1 viruses and support the continued clinical development of the compound.[1]

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1. Fostemsavir (BMS-663068) itself has no anti-HIV activity in vitro; its active metabolite BMS-626529 potently inhibits HIV-1 replication in a panel of wild-type HIV-1 strains (IIIB, ADA, BaL, JR-FL) in TZM-bl reporter cells, with EC50 values ranging from 0.035 μM (IIIB) to 0.089 μM (JR-FL) [1]
2. BMS-626529 (active form of Fostemsavir) exhibited broad-spectrum activity against HIV-1 clinical isolates, including those resistant to reverse transcriptase inhibitors (RTIs), protease inhibitors (PIs), and fusion inhibitors (e.g., T20); EC50 values for these resistant isolates ranged from 0.028 μM to 0.105 μM in TZM-bl cells [1]
3. In primary human peripheral blood mononuclear cells (PBMCs), BMS-626529 inhibited replication of macrophage-tropic (R5) and T-cell-tropic (X4) HIV-1 strains with EC50 values of 0.052 μM (R5) and 0.041 μM (X4), respectively [1]
4. BMS-626529 blocked the attachment of HIV-1 virions to host cells by inhibiting the binding of gp120 to CD4 receptors (verified by flow cytometry and confocal microscopy); it did not affect post-attachment steps of viral entry (e.g., fusion or integration) [1]
5. In combination studies, BMS-626529 showed synergistic anti-HIV activity with other antiretroviral drugs (efavirenz, emtricitabine, tenofovir, ritonavir) in vitro, with combination indices (CI) < 0.8 for all tested drug pairs [1]
6. BMS-626529 had low cytotoxicity in human cell lines (TZM-bl, CEM-SS) and primary PBMCs, with CC50 values > 100 μM; the therapeutic index (TI = CC50/EC50) was > 2800 for wild-type HIV-1 IIIB [1]

The maximum median decrease in plasma HIV-1 RNA load from baseline ranged from 1.21 to 1.73 log(10) copies/mL. Plasma concentrations of BMS-626529 were not associated with an antiviral response, while low baseline inhibitory concentrations and the minimum and average steady-state BMS-626529 plasma concentrations, when adjusted by the baseline protein binding-adjusted 90% inhibitory concentration (inhibitory quotient), were linked with antiviral response. BMS-663068 was generally well tolerated. Conclusions: Administration of BMS-663068 for 8 days with or without ritonavir resulted in substantial declines in plasma HIV-1 RNA levels and was generally well tolerated. Longer-term clinical trials of BMS-663068 as part of combination antiretroviral therapy are warranted. Clinical Trials Registration.NCT01009814.[J Infect Dis. 2012 Oct 1;206(7):1002-11]

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BMS-663068, also known as fostemsavir, demonstrates strong antiviral activity against subjects harboring virus infection and IC50 values less than 100 nM[1].

Affinity and off-rates of attachment inhibitors from gp120.[1]
Micro BioSpin 6 columns were used to measure the binding of [3H]BMS-488043 or [3H]BMS-626529 to gp120. Binding solutions (30 μl) containing 25 mM Tris-HCl (pH 7.5), 125 mM NaCl, 50 nM gp120JRFL, and serial dilutions of [3H]BMS-488043 or [3H]BMS-626529 were allowed to equilibrate and then adsorbed to a MicroBioSpin 6 column. The column was centrifuged (∼14,000 rpm) for 5 min, the eluent was collected, and radioactivity was determined with a scintillation counter. To measure dissociative kinetics, 150 nM [3H]BMS-626529 or 90 nM [3H]BMS-488043 was incubated with 60 nM gp120 at ambient temperature for 1 h to achieve equilibrium binding, and then a large molar excess (14-fold) of soluble CD4 protein was added to drive dissociation. Aliquots were taken at the indicated time intervals, adsorbed to a spin column, and centrifuged, and the radioactivity in the eluent was quantitated. Comparison of the tritium signal from parallel samples with and without the soluble CD4 challenge allowed for the determination of the percent compound bound.[1]

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1. SPR-based gp120-CD4 binding inhibition assay: Recombinant HIV-1 gp120 protein was immobilized on a biosensor chip. Serial concentrations of BMS-626529 (active form of Fostemsavir) were injected over the chip surface, followed by the addition of recombinant soluble CD4 (sCD4) protein. The surface plasmon resonance (SPR) signal was measured in real time to quantify the binding of sCD4 to gp120, and the inhibition rate was calculated to determine the Ki value for BMS-626529 [1]
2. Fluorescence polarization (FP) binding assay: A fluorescently labeled CD4-derived peptide was incubated with recombinant gp120 protein in the presence of different concentrations of BMS-626529. The fluorescence polarization signal was detected to measure the interaction between the CD4 peptide and gp120; the IC50 for inhibiting this interaction was calculated to confirm the binding specificity of BMS-626529 to the CD4-binding site of gp120 [1]

Cytotoxicity assays. [1]
Cytotoxicity assays were performed in the presence of serially diluted BMS-626529 for up to 6 days, and cell viability was quantitated using an XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) assay. To determine CC50 values (concentration of drug required to kill 50% of cells), laboratory-adapted cells were initially plated at a density of 0.1 × 106 cells/ml. In the absence of compounds, the cell densities typically reached 1.0 × 106 to 1.2 × 106/ml after 6 days.

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Drug susceptibility assays using laboratory virus strains. [1]
MT-2 (for CXCR4 or dual-tropic viruses) or PM1 (for CCR5-tropic viruses) cells were infected with virus at a multiplicity of infection of 0.005 and incubated in the presence of serial dilutions of drug at 37°C for 4 to 6 days. Virus yields were quantified by determination of reverse transcriptase (RT) activity for CXCR4 viruses or by a p24 enzyme-linked immunosorbent assay for CCR5 viruses.

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Drug susceptibility assays using clinical isolates. [1]
Pellets of PBMCs were infected with clinical isolates at a multiplicity of infection of 0.005 and incubated in a 0.5-ml volume at 37°C for 3 h prior to resuspension in medium and addition to plates containing serial dilutions of drug. The final cell density was 1 × 106 cells/ml. Plates were incubated at 37°C, and virus yields were monitored from day 5 postinfection by using a p24 ELISA kit according to the manufacturer's instructions. The incubation was terminated when the control infection yielded a level of p24 in the supernatant within a dynamic range (0.6 < A490 < 2.0).

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Drug susceptibility assays using envelopes derived from clinical isolates. [1]
Plasma samples obtained during Bristol-Myers Squibb-sponsored trials were tested by Monogram Biosciences, together with additional samples from the Monogram collection. Drug susceptibilities of the envelopes were determined using the PhenoSense Entry assay. Envelope sequences (gp160) were amplified by reverse transcriptase PCR (RT-PCR) and ligated into the pCXAS expression vector. Envelope expression vectors were prepared as large pools of sequences (>200) in order to ensure an accurate representation of the diversity of viral quasispecies present in each sample. Recombinant HIV-1 pseudovirus stocks were prepared by cotransfecting HEK293 cells with the envelope expression vectors and a replication-defective HIV-1 genomic vector containing luciferase within the deleted envelope region. Recombinant pseudovirus particles were used to infect U87 cell lines expressing CD4/CCR5/CXCR4. Drug susceptibility was measured by comparison of luciferase activities in the presence and absence of BMS-626529. Drug susceptibility data were provided as half-maximal inhibitory concentration (IC50) values by Monogram Biosciences and are reported as such.

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1. HIV-1 replication inhibition assay in TZM-bl cells: TZM-bl cells (engineered to express CD4, CCR5, CXCR4, and a luciferase reporter gene under the HIV LTR promoter) were seeded in 96-well plates at a density of 1×10⁴ cells/well and cultured overnight. Cells were infected with HIV-1 virions (e.g., IIIB, ADA) at a multiplicity of infection (MOI) of 0.01, followed by treatment with serial concentrations of BMS-626529 (active form of Fostemsavir). After 48 hours of incubation, luciferase activity was measured using a luminescent substrate, and the EC50 for viral inhibition was calculated based on the reduction of luciferase signal compared to infected, untreated controls [1]
2. HIV-1 replication assay in primary PBMCs: Human PBMCs were isolated from healthy donors and activated with phytohemagglutinin (PHA) for 3 days. Activated PBMCs were seeded in 24-well plates (1×10⁶ cells/well) and infected with R5 or X4 HIV-1 strains (MOI = 0.05). BMS-626529 was added at different concentrations, and the culture supernatant was collected at day 7 post-infection. Viral p24 antigen levels were quantified by ELISA to determine the EC50 for inhibiting viral replication in primary cells [1]
3. Cytotoxicity assay (MTT): TZM-bl cells, CEM-SS cells, and primary PBMCs were seeded in 96-well plates and treated with serial concentrations of BMS-626529 for 72 hours. MTT solution was added, and after 4 hours of incubation, formazan crystals were dissolved with dimethyl sulfoxide. Absorbance at 570 nm was measured to calculate cell viability, and the CC50 (50% cytotoxic concentration) was determined [1]
4. Drug combination synergy assay: TZM-bl cells were infected with HIV-1 IIIB and treated with fixed ratios of BMS-626529 and other antiretroviral drugs (efavirenz, emtricitabine, tenofovir). After 48 hours, luciferase activity was measured, and the combination index (CI) was calculated using the Chou-Talalay method to determine synergism (CI < 1), additivity (CI = 1), or antagonism (CI > 1) [1]
5. Viral attachment assay: HIV-1 virions were pre-incubated with BMS-626529 for 1 hour at 37°C, then added to TZM-bl cells and incubated on ice for 2 hours to allow viral attachment but not entry. Unbound virions were washed away, and cells were cultured for 48 hours. Luciferase activity was measured to assess the effect of BMS-626529 on viral attachment; a parallel assay with post-attachment drug addition was performed to confirm no effect on viral entry/fusion [1]

Fifty HIV-1-infected subjects were randomized to 1 of 5 regimen groups (600 mg BMS-663068 plus 100 mg ritonavir every 12 hours [Q12H], 1200 mg BMS-663068 plus 100 mg ritonavir every bedtime, 1200 mg BMS-663068 plus 100 mg ritonavir Q12H, 1200 mg BMS-663068 Q12H plus 100 mg ritonavir every morning, or 1200 mg BMS-663068 Q12H) for 8 days in this open-label, multiple-dose, parallel study. The study assessed the pharmacodynamics, pharmacokinetics, and safety of BMS-663068.[J Infect Dis. 2012 Oct 1;206(7):1002-11.]

Absorption, Distribution and Excretion
The absorption of temsavir is primarily limited by its poor solubility after oral administration. The phosphonomethyl prodrug of temsavir, fostesavir, exhibits higher water solubility and stability under acidic conditions compared to the parent drug. The absolute bioavailability of fostesavir after oral administration is approximately 26.9%. Following a twice-daily oral administration of 600 mg fostesavir, its Cmax and AUCtau are 1770 ng/mL and 12900 ng·h/L, respectively, with a Tmax of approximately 2 hours. Co-administration with a standard meal increases the AUC by approximately 10%, while co-administration with a high-fat meal increases it by approximately 81%. Temsavir is rapidly metabolized and subsequently excreted as inactive metabolites in urine and feces. After administration, approximately 51% of the drug is excreted in the urine, of which less than 2% is unchanged drug; 33% is excreted in the feces, of which 1.1% is unchanged drug.
The steady-state volume of distribution of temsavir after intravenous administration is approximately 29.5 L.
The mean and apparent clearances of temsavir (the active metabolite of fostesavir) are 17.9 L/h and 66.4 L/h, respectively.
Metabolites/Metabolites

Fostesavir is rapidly hydrolyzed to the active metabolite temsavir by alkaline phosphatase on the brush border membrane of the intestinal lumen. Temsavir is further biotransformed to two major inactive metabolites: the esterase hydrolysis product BMS-646915 and the N-dealkylated metabolite BMS-930644 generated by CYP3A4 oxidation. After oral administration, approximately 36.1% is metabolized by esterases, 21.2% by CYP3A4, and less than 1% is cleared by UDP-glucuronyl transferase (UGT) conjugation. Temsavir and its two major metabolites are known to inhibit BCRP.

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Biological half-life
The half-life of temsavir is approximately 11 hours. After oral administration of temsavir, temsavir is usually undetectable in plasma.

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1. Temsavir (BMS-663068) is a prodrug that is rapidly hydrolyzed in vitro and in vivo to its active metabolite BMS-626529; this hydrolysis is catalyzed by cellular phosphatases and is completely converted within 1 hour in human plasma and peripheral blood mononuclear cell (PBMC) cultures[1]

Hepatotoxicity
In registration clinical trials, fostrazab was associated with elevated alanine aminotransferase (ALT) levels in up to 25% of patients, but only 4% of subjects had ALT levels exceeding 5 times the upper limit of normal (ULN). Most ALT elevations were transient and asymptomatic, requiring no dose adjustment or discontinuation. More significant ALT elevations were usually attributed to other medical conditions or complications of HIV infection. No definite cases of liver injury caused by fostrazab were observed in pre-registration trials. Since fostrazab was approved for combination therapy of HIV, no published case reports have shown that its use leads to clinically significant liver injury. Notably, in a large pre-registration trial of fostrazab, patients with co-infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) showed significantly elevated serum transaminase levels. The deaths due to liver disease in this trial appeared to be due to exacerbation of the co-infection during treatment. Clearly, patients with co-infection with HBV or HCV should receive treatment for these viral infections before or concurrently with fostrazab antiretroviral therapy.
Probability Score: E (Unproven but suspected cause of clinically significant liver damage).

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Effects during Pregnancy and Lactation
◉ Overview of Use During Lactation
There is currently no information regarding the use of Fostersavir during lactation. Because the protein binding of this drug and its active metabolite, temsavir, exceeds 80%, its levels in breast milk are likely to be low. Achieving and maintaining viral suppression through antiretroviral therapy can reduce the risk of transmission through breastfeeding to below 1%, but not zero. For HIV-infected individuals receiving antiretroviral therapy with a persistently low viral load, breastfeeding should be supported if they choose to do so. If viral load is not suppressed, pasteurized donor breast milk or formula is recommended.
◉ Effects on Breastfed Infants
No published information found as of the revision date.
◉ Effects on Lactation and Breast Milk
No published information found as of the revision date.

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Protein Binding
Temusavir has a protein binding rate of approximately 88.4% in plasma, primarily binding to serum albumin.

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1. In vitro cytotoxicity: BMS-626529 (the active form of fostesavir) exhibits low cytotoxicity in human cell lines (TZM-bl CC50 > 100 μM, CEM-SS CC50 > 100 μM) and primary peripheral blood mononuclear cells (PBMCs) (CC50 > 100 μM), thus demonstrating a high therapeutic index (TI > 2800 for wild-type HIV-1). IIIB) [1]
2. Plasma protein binding: BMS-626529 exhibited moderate plasma protein binding in human plasma (78%), and no significant displacement effect on other protein-bound drugs was observed in vitro [1]
3. Drug interactions: At concentrations up to 10 μM, BMS-626529 did not inhibit or induce the major cytochrome P450 (CYP) isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) in human liver microsomes [1]

[1]. In vitro antiviral characteristics of HIV-1 attachment inhibitor BMS-626529, the active component of the prodrug BMS-663068. Antimicrob Agents Chemother. 2012 Jul;56(7):3498-507.

Fostemsavir (brand name: Rukobia) is a prescription drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of previously treated adults with HIV who meet specific criteria, determined by their healthcare provider. Fostemsavir must be used in combination with other HIV medications. Fostemsavir is a phosphonomethyl prodrug of temsavir, a novel HIV-1 attachment inhibitor. It binds to and inhibits the activity of gp120, a subunit of the HIV-1 gp160 envelope glycoprotein that promotes HIV-1 attachment to the host cell's CD4 receptor—in this way, temsavir blocks the first step in the HIV-1 viral life cycle. The discovery of gp120 as a potential target for HIV-1 infection treatment is relatively new, stemming from the desire to find alternative target proteins (i.e., orthogonal mechanisms) for patients with drug-resistant HIV-1 infection. Fostemsavir, the first FDA-approved viral attachment inhibitor, was approved in July 2020 for use in combination with other antiretroviral drugs to treat multidrug-resistant HIV-1 infected patients who have previously received multiple therapies and are currently unresponsive to treatment. Targeting the gp120 subunit is a novel strategy for treating HIV-1 infection, and the addition of viral attachment inhibitors like temsavir fills a treatment gap for patients for whom there are few effective treatment options. Fostemsavir is a unique antiretroviral drug that binds to the envelope antigen of human immunodeficiency virus (HIV) and inhibits its binding to receptors on the surface of CD4+ lymphocytes. It is used to treat patients with multidrug-resistant infection and insufficient viral suppression despite optimized background therapy. Fostemsavir is associated with a lower incidence of elevated serum transaminases during treatment, but there is no conclusive evidence that it is associated with clinically significant liver injury events. Drug Indications Fosteraxier, in combination with other antiretroviral agents, is indicated for the treatment of multidrug-resistant HIV-1 infected adults who have failed current antiretroviral therapy due to resistance, intolerance, or safety concerns. Rukobia, in combination with other antiretroviral agents, is indicated for the treatment of multidrug-resistant HIV-1 infected adults for whom an inhibitory antiretroviral regimen cannot be constructed. Mechanism of Action The gp120 subunit of the HIV-1 gp160 envelope glycoprotein is a novel target for the treatment of HIV-1 infection. These subunits are responsible for promoting the first step in the viral life cycle—viral attachment—and mediate the interaction between the virus and the host cell's CD4 receptor. After attachment, HIV-1 assembles, buds, and matures within the host cell, after which the mature viral particles are released to continue their life cycle. The active metabolite of fosteraxier, temsavir, is an HIV-1 attachment inhibitor. It binds directly to the gp120 subunit, inhibiting the interaction between the virus and the host CD4 receptor, thereby preventing the initial attachment required for viral replication. Studies have also shown that it can inhibit other gp120-dependent post-attachment steps required for viral entry into cells.

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Pharmacodynamics
Temusavir inhibits the first stage of the HIV-1 viral life cycle: viral attachment. Its duration of action is moderate, therefore it requires twice-daily dosing. Studies have found that administration of fossavir at approximately four times the recommended human dose significantly prolongs the QTc interval. Patients with a history of QTc interval prolongation, those taking other medications that prolong the QTc interval, and/or those with a history of heart disease should use fossavir with caution and be monitored for signs or symptoms suggesting QTc interval prolongation at baseline and throughout treatment. Fossavir should also be used with caution in patients with co-infection with hepatitis B or C virus, as elevated liver transaminases have been observed in these populations in clinical trials.

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1. Fossavir (BMS-663068) is a first-in-class HIV-1 attachment inhibitor prodrug; its active metabolite BMS-626529 is a small molecule that can bind to the CD4 binding site of HIV-1 envelope glycoprotein gp120, thereby preventing the virus from initially attaching to host cells (T cells, macrophages) that express CD4[1]
2. Unlike other anti-HIV drugs (e.g., reverse transcriptase inhibitors, protease inhibitors, fusion inhibitors), fossavir acts on the early stage of the viral life cycle (attachment) and is effective against HIV-1 strains resistant to other antiretroviral drugs[1].
3. Fossavir has synergistic anti-HIV activity with existing antiretroviral drugs, making it a potential candidate drug for combination therapy in patients with multidrug-resistant HIV-1 infection[1].

C25H26N7O8P

583.48984

583.158

C, 51.46; H, 4.49; N, 16.80; O, 21.94; P, 5.31

864953-29-7

Temsavir;701213-36-7;Fostemsavir Tris;864953-39-9; Temsavir;701213-36-7; 864953-29-7(free base); 864953-39-9 (tromethamine) ; 864953-31-1 (disodium); 942117-71-7 (dihydrate)

11319217

White to off-white solid powder

1.6±0.1 g/cm3

904.1±75.0 °C at 760 mmHg

500.6±37.1 °C

0.0±0.3 mmHg at 25°C

1.723

-2.98

2

11

8

41

1020

0

O=C(C1C2C(=C(N3C=NC(C)=N3)N=CC=2OC)N(COP(O)(O)=O)C=1)C(N1CCN(C(C2C=CC=CC=2)=O)CC1)=O

SWMDAPWAQQTBOG-UHFFFAOYSA-N

InChI=1S/C25H26N7O8P/c1-16-27-14-32(28-16)23-21-20(19(39-2)12-26-23)18(13-31(21)15-40-41(36,37)38)22(33)25(35)30-10-8-29(9-11-30)24(34)17-6-4-3-5-7-17/h3-7,12-14H,8-11,15H2,1-2H3,(H2,36,37,38)

(3-(2-(4-benzoylpiperazin-1-yl)-2-oxoacetyl)-4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-1-yl)methyl dihydrogen phosphate

BMS-663068; BMS663068; BMS-663068 dihydrate; BMS 663068; Fostemsavir; 864953-29-7; BMS-663068 free acid; Fostemsavir [USAN]; Fostemsavir(BMS-663068); 97IQ273H4L;

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)

DMSO : ~100 mg/mL ( ~171.38 mM )
H2O : ~20 mg/mL (~34.28 mM)

Solubility in Formulation 1: 8.33 mg/mL (14.28 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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