Some info on IGF and MMD

1: J Clin Endocrinol Metab. 1995 Dec;80(12):3715-23.

Metabolic and clinical response to recombinant human insulin-like growth factor
I in myotonic dystrophy--a clinical research center study.

Vlachopapadopoulou E, Zachwieja JJ, Gertner JM, Manzione D, Bier DM, Matthews
DE, Slonim AE.

Department of Pediatrics, New York Hospital-Cornell Medical Center, New York
10021, USA.

Muscle weakness and wasting in myotonic dystrophy (MyD) are believed to be due
to a decrease in muscle protein synthesis, secondary to insulin resistance. A
4-month, randomized, double blind, placebo-controlled trial was undertaken to
assess whether recombinant human insulin-like growth factor I (rhIGF-I) may
overcome the insulin resistance. Patients received either 5 mg rhIGF-I (n = 7)
or placebo (n = 9), sc, twice daily. Glucose metabolism was assessed by stable
label iv glucose tolerance test, amino acid metabolism by L-[13C] leucine
turnover, body composition by dual energy x-ray absorptiometry and N excretion,
and muscle response by manual muscle strength and neuromuscular function. In the
treated group, the insulin sensitivity index, insulin action, and glucose
disposal all increased (P < 0.05). Leucine flux and leucine incorporation into
protein increased (P < 0.05), and the rate of leucine oxidation to leucine
turnover decreased (P < 0.05), findings indicative of increased protein
synthesis. Body weight and lean body mass increased, whereas percent body fat
decreased (P < 0.05). An increase in manual muscle strength of 0.42 +/- 0.30 (P
< 0.02) and in neuromuscular function of 17.5 +/- 11.7 (P < 0.02) occurred in
the four patients who received a rhIGF-I dose greater than 70 micrograms/kg,
whereas a more modest response occurred in the three patients who received a
dose less than 70 micrograms/kg. Two patients showed dramatic improvement. Long
term rhIGF-I therapy appears to cause metabolic and muscle improvement in
treated MyD patients.

2. Endocrinology. 1999 Sep;140(9):4244-50.

Insulin-like growth factor I circumvents defective insulin action in human
myotonic dystrophy skeletal muscle cells.

Furling D, Marette A, Puymirat J.

Department of Medicine, Laval University Medical Research Center, University
Hospital Center of Quebec, Canada.

Primary human skeletal muscle cell cultures derived from muscles of a myotonic
dystrophy (DM) fetus provided a model in which both resistance to insulin action
described in DM patient muscles and the potential ability of insulin-like growth
factor I (IGF-I) to circumvent this defect could be investigated. Basal glucose
uptake was the same in cultured DM cells as in normal myotubes. In DM cells, a
dose of 10 nM insulin produced no stimulatory effect on glucose uptake, and at
higher concentrations, stimulation of glucose uptake remained significantly
lower than that in normal myotubes. In addition, basal and insulin-mediated
protein synthesis were both significantly reduced compared with those in normal
cells. In DM myotubes, insulin receptor messenger RNA expression and insulin
receptor binding were significantly diminished, whereas the expression of GLUT1
and GLUT4 glucose transporters was not affected. These results indicate that
impaired insulin action is retained in DM cultured myotubes. The action of
recombinant human IGF-I (rhIGF-I) was evaluated in this cellular model. We
showed that rhIGF-I is able to stimulate glucose uptake to a similar extent as
in control cells and restore normal protein synthesis level in DM myotubes.
Thus, rhIGF-I is able to bypass impaired insulin action in DM myotubes. This
provides a solid foundation for the eventual use of rhIGF-I as an effective
treatment of muscle weakness and wasting in DM.


3: Am J Clin Nutr. 2004 Aug;80(2):357-64.

Postabsorptive and insulin-stimulated energy and protein metabolism in patients
with myotonic dystrophy type 1.

Perseghin G, Comola M, Scifo P, Benedini S, De Cobelli F, Lanzi R, Costantino F,
Lattuada G, Battezzati A, Del Maschio A, Luzi L.

Internal Medicine Section of Nutrition/Metabolism, Universita Vita e Salute San
Raffaele, Istituto Scientifico H San Raffaele, via Olgettina 60, 20132 Milan,
Italy. perseghin.gianluca@hsr.it

BACKGROUND: Exaggerated insulin resistance was described as the major metabolic
abnormality in myotonic dystrophy type 1 (DM1). We reported recently that the
severity of the impairment in insulin-stimulated glucose metabolism in these
patients was overestimated. OBJECTIVE: The aim was to dissect out insulin action
with respect to whole-body energy homeostasis and glucose, protein, and lipid
metabolism in patients with DM1 to assess the relevance of insulin resistance to
the heterogeneous clinical manifestations of this syndrome. DESIGN: Ten
nondiabetic patients with DM1 and 10 matched healthy control subjects were
studied by means of 1) dual-energy X-ray absorptiometry; 2) a
euglycemic-hyperinsulinemic clamp (40 mU. m(-2). min(-1)) combined with a
primed, continuous infusion of [6,6-d(2)]glucose and [1-(13)C]leucine; 3)
indirect calorimetry; and 4) localized (1)H magnetic resonance spectroscopy of
the calf muscles. RESULTS: Patients with DM1 had less lean body mass, greater
fat mass, and greater intramyocellular lipid contents than did healthy control
subjects. Energy expenditure and glucose and lipid metabolism did not differ
significantly between the groups. In contrast, markers of proteolysis were
higher in DM1 patients in the postabsorptive and insulin-stimulated conditions
and were associated with lower plasma concentrations of insulin-like growth
factor 1 (P < 0.03) and higher plasma concentrations of tumor necrosis factor
alpha receptor 2 (P = 0.04). CONCLUSIONS: Despite greater body fat and
intramyocellular lipid contents in patients with DM1, insulin sensitivity was
not significantly different between patients and control subjects. In contrast,
the loss of lean body mass in patients with DM1 was associated with abnormal
postabsorptive and insulin-stimulated regulation of protein breakdown. Lower
plasma insulin-like growth factor 1 concentrations and higher tumor necrosis
factor system activity might be involved in the muscle wasting of DM1.

The method used by Phyton utilizes transformed plant cells instead of bacterial cells (E. coli) to generate the protein of interest (IGF in this case). The bacterial approach is used everyday by most labs in the country to generate protein of interest. It is a very simple ans standard methodology. It appears that Phyton specializes in production of protein via plant cell host. Since this is their area of specialty, it should not be difficult for them to apply same methodology to produce large scale of IGF or any other protein of interest. FDA will not require new trial. They will however will want to verify purity of the product and the commercial plant fromwhich IGF is produced. IMHO, it comes down to revenues and profit- if Phyton will be rewarded appropriately for their efforts, they will be able to fulfill the needs of INSM relatively quickly- question is how much is INSM willing to share with Phyton.

IMHO, this is a potentially very important development for INSM. It is a clearly an attempt to circumvent the TRCA patent infringement lawsuit. The lawsuit clearly states that INSM violated the DNA patent which describes IGF production via prokayotic host (bacterial cell). Phyton will produce IGF via plant cell host (eukaryotic cell). This will completely nullify TRCA lawsuit- in the event that TRCA wins the lawsuit. If successful, this is a brilliant move and will eliminate the patent infringement concerns. Overall, this is a very positive development.

Following are the abstracts of those papers: As I mentioned the Patent TRCA calims as their novel method is a very simple method that has been used for years and is taught in every first year molecular biology textbook. As you can see, the first description of the same method as Patent 414 was published in 1985. Ther is no way this patent can hold. However, having said this, INSM needs to hire better lawyers, their preparation and defense during the last hearing was terrible.


1: J Mol Endocrinol. 1992 Jun;8(3):213-23.

Novel recombinant fusion protein analogues of insulin-like growth factor (IGF)-I
indicate the relative importance of IGF-binding protein and receptor binding for
enhanced biological potency.

Francis GL, Ross M, Ballard FJ, Milner SJ, Senn C, McNeil KA, Wallace JC, King
R, Wells JR.

Cooperative Research Centre for Tissue Growth and Repair, CSIRO Division of
Human Nutrition, Adelaide, South Australia.

An efficient expression system in Escherichia coli for several biologically
active insulin-like growth factor-I (IGF-I) fusion peptide analogues is
described. These novel IGF-I fusion protein analogues have properties that make
them very useful reagents in the investigation of IGF-I action. The analogues
comprise an IGF-I sequence and the first 11 amino acids of methionyl porcine
growth hormone (pGH) and include [Met1]-pGH(1-11)-Val-Asn-IGF-I, which contains
the authentic IGF-I sequence, and two analogues,
[Met1]-pGH(1-11)-Val-Asn-[Gly3]-IGF-I and [Met1]-pGH(1-11)-Val-Asn-[Arg3]-IGF-I,
where Glu-3 in the human IGF-I sequence has been replaced by Gly or Arg
respectively. The three peptides are referred to as Long IGF-I, Long
[Gly3]-IGF-I or Long [Arg3]-IGF-I depending on the IGF-I sequence present.
Production of the purified fusion peptides was aided by folding the reduced and
denatured fusion peptide sequence under conditions that gave very high yields of
biologically active product. Introduction of a hydrophobic N-terminal extension
peptide appears to facilitate the correct folding of the IGF-I analogues
compared with that obtained previously when folding normal-length IGFs. The
biological activities of the IGF-I fusion peptides were compared with authentic
IGF-I and the truncated analogue, des(1-3)IGF-I. In L6 rat myoblasts, all the
analogues were more potent than authentic IGF-I in their abilities to stimulate
protein and DNA synthesis and inhibit protein breakdown. In H35 hepatoma cells,
where the IGFs act through the insulin receptor, the Long IGF-I analogues
maintained a similar potency relative to IGF-I as was observed in the L6
myoblasts. The order of biological potency in cell lines secreting IGF-binding
proteins (IGFBPs) into the medium was Long [Arg3]-IGF-I-des(1-3)IGF-I greater
than Long [Gly3]-IGF-I greater than Long IGF-I greater than IGF-I. In chicken
embryo fibroblasts, a cell line that does not secrete detectable IGFBPs into the
medium, Long [Arg3]-IGF-I, was less potent than IGF-I. Investigation of receptor
and IGFBP association by these analogues reinforced our previous findings that
N-terminal analogues of IGF-I show increased biological potency due to changes
in the degree of their IGFBP interactions.

PMID: 1378742 [PubMed - indexed for MEDLINE]

2: J Mol Endocrinol. 1992 Feb;8(1):29-41.

Production and characterization of recombinant insulin-like growth factor-I
(IGF-I) and potent analogues of IGF-I, with Gly or Arg substituted for Glu3,
following their expression in Escherichia coli as fusion proteins.

King R, Wells JR, Krieg P, Snoswell M, Brazier J, Bagley CJ, Wallace JC, Ballard
FJ, Ross M, Francis GL.

Department of Biochemistry, University of Adelaide, South Australia.

The development of an efficient expression system for insulin-like growth
factor-I (IGF-I) in Escherichia coli as a fusion protein is described. The
fusion protein consists of an N-terminal extension made up of the first 46 amino
acids of methionyl porcine GH ([Met1]-pGH) followed by the dipeptide Val-Asn.
The latter two residues provide a unique hydroxylamine-sensitive link between
[Met1]-pGH(1-46) and the N-terminal Gly of IGF-I. Downstream processing of the
fusion proteins involved isolation of inclusion bodies, cleavage at the Asn-Gly
bond, refolding of the reduced IGF-I peptide and purification to homogeneity.
This expression system was also used to produce two variants of IGF-I in which
Glu3 was substituted by either Gly or Arg to give [Gly3]-IGF-I and [Arg3]-IGF-I
respectively. Production of milligram quantities of IGF-I peptide was readily
achieved. The purity of the IGF-I, [Gly3]-IGF-I and [Arg3]-IGF-I was established
by high-performance liquid chromatography and N-terminal sequence analysis.
[Gly3]-IGF-I and [Arg3]-IGF-I were more potent than IGF-I in biological assays
measuring stimulation of protein synthesis and DNA synthesis or inhibition of
protein breakdown in rat L6 myoblasts. Both analogues bound very poorly to
bovine IGF-binding protein-2 and slightly less well than IGF-I to the type-1
receptor on rat L6 myoblasts. We conclude that reduced binding to IGF-binding
proteins rather than increased receptor binding is the likely explanation for
the greater biological potency of the analogues compared with IGF-I.

PMID: 1311930 [PubMed - indexed for MEDLINE]

3: Methods Enzymol. 1991;198:3-16.

Expression and purification of recombinant insulin-like growth factors from
Escherichia coli.

Nilsson B, Forsberg G, Hartmanis M.

PMID: 1857223 [PubMed - indexed for MEDLINE]

4: Eur J Biochem. 1989 Apr 1;180(3):555-61.

Gene synthesis, expression in Escherichia coli and purification of
immunoreactive human insulin-like growth factors I and II. Application of a
modified HPLC separation technique for hydrophobic proteins.

Hummel M, Herbst H, Stein H.

Institute of Pathology, Klinikum Steglitz, Free University of Berlin.

We have expressed synthetic genes encoding human insulin-like growth factors I
and II in large quantities in Escherichia coli as fusion proteins with the 300
N-terminal amino acids of the E. coli trpE gene product. The resulting hybrid
proteins were purified from the insoluble fraction of crude bacterial lysates
using a rapid HPLC separation procedure employing a C8 reversed-phase column and
a gradient of 2-propanol in formic acid. With the procedure we obtained in
volatile solvents highly purified fusion proteins that were used for further
biochemical and immunological procedures. Here we describe biochemical
characteristics of the bacterially expressed fusion proteins and demonstrate
that these proteins share substantial antigenic properties with the native
polypeptides allowing the establishment of highly specific monoclonal
antibodies.

PMID: 2469575 [PubMed - indexed for MEDLINE]

5: Gene. 1988 Sep 7;68(2):193-203.

Expression of secreted insulin-like growth factor-1 in Escherichia coli.

Wong EY, Seetharam R, Kotts CE, Heeren RA, Klein BK, Braford SR, Mathis KJ,
Bishop BF, Siegel NR, Smith CE, et al.

Department of Biological Sciences, Monsanto Co., Chesterfield, MO 63198.

The synthesis, processing and secretion of insulin-like growth factor-1 (IGF-1
or somatomedin-C) fused to LamB and OmpF secretion leader sequences in
Escherichia coli have been investigated. Expression and secretion of IGF-1 was
achieved. The major portion of this secreted IGF-1 accumulated in the
periplasmic space as insoluble aggregates. A small amount of IGF-1 was found
folded in its native conformation in the medium. The lamB and ompF signal
sequences were fused to the 5' coding sequence of IGF-1. Fusion of the lamB
signal sequence directly to IGF-1 (lamB-IGF-1) resulted in accumulation of 16-20
micrograms/A550/ml of correctly processed IGF-1 in the periplasmic space. The
processing efficiency of LamB-IGF-1 and OmpF-IGF-1 was enhanced in an E. coli
strain bearing a prlA4 mutation. Amino acid sequence analysis of IGF-1 secreted
into the periplasm and exported into the medium confirmed the precise removal of
the LamB or OmpF signal sequence. IGF-1 synthesized in E. coli was demonstrated
to be active in a cell proliferation bioassay.

PMID: 3065142 [PubMed - indexed for MEDLINE]

6: Biochemistry. 1987 Aug 25;26(17):5239-44.

Expression of human insulin-like growth factor I in bacteria: use of optimized
gene fusion vectors to facilitate protein purification.

Moks T, Abrahmsen L, Holmgren E, Bilich M, Olsson A, Uhlen M, Pohl G, Sterky C,
Hultberg H, Josephson S, et al.

Department of Biochemistry, Royal Institute of Technology, Stockholm, Sweden.

Several fusions between the gene for human insulin-like growth factor I (IGF-I)
and the genes for different IgG-binding fragments of staphylococcal protein A
were assembled and compared regarding expression, secretion, and purification of
the peptide hormone. After IgG affinity purification of the fusion proteins from
the growth medium of Staphylococcus aureus or Escherichia coli, native IGF-I was
released by cleavage of an Asn-Gly peptide bond with hydroxylamine. An optimized
expression system based on a modified synthetic IgG-binding domain (z),
resistant to hydroxylamine, gave the highest yield of fusion protein. After
cleavage, the hormone could be separated from the IgG-binding moiety and from
noncleaved fusion protein by a second passage through the IgG affinity column.
The biological activity and the purity of the IGF-I obtained were confirmed by a
radioreceptor assay, N-terminal sequence analysis, polyacrylamide gel
electrophoresis, isoelectric focusing, and high-performance liquid
chromatography.

PMID: 3676250 [PubMed - indexed for MEDLINE]

7: Nucleic Acids Res. 1985 Mar 25;13(6):1923-38.

Optimizing the expression in E. coli of a synthetic gene encoding somatomedin-C
(IGF-I).

Buell G, Schulz MF, Selzer G, Chollet A, Movva NR, Semon D, Escanez S, Kawashima
E.

Double-stranded DNA encoding the human hormone somatomedin-C (SMC) has been
synthesized. This synthetic gene has been inserted into a plasmid bearing the
strong leftward promoter (PL) of bacteriophage lambda and expressed in E. coli.
Codons for the N-terminal region of SMC which maximized the hormone's synthesis
were chosen in an SMC-lac z fusion assay. The amounts of SMC accumulated in E.
coli were influenced by mutations at two chromosomal loci, lon and htpR.

Previously I posted the following- shortly after the initial decision was rendered:


"When I read the decision what I was struck with was the broadness and non-specificness of the patents. As this initial decision is just setting the ground rules, there are lot more twists and turns to come.

My initial impression was TRCA fared much better than INSM- which probably was the case. It appeared from the transcript that TRCA was better prepared than INSM. After having thought about this for most of the day (after being upset and down most of the day), I really feel that this initial decision was a non-event. The real trial is yet to come- nothing was settled here.

In reading the decision, it is quite apparent that the patents were broad as possible and I (not being a lawyer but a physician scientist) can already see many potential weaknesses with the position taken by TRCA. First, the patent for the production of IGF is as broad as can be- including usage of all prokaryotic host (not just E. coli). There are number of very broad application of the process that are mentioned throughout. There were some areas that the judge also had raised some issues for TRCA. The brief summary by Unterberg previously posted also provides some insight. SOme things that bother me about TRCA patent position are 1) the broadness of the patents in regards to production -they cover all prokaryotes (all bacterial hosts), 2) the broadness or nonspecific definition of IGF, fusion protein and etc..., 3) the method they described for IGF production is not unique or novel but a simple standard molecular methodology that is published in any first year college molecular biology textbook- it completely baffles me how this can be considered a patent but what do I know, 4) judge in this case just laid down the groundrules but did not entertain arguments as to why the patents are invalid- I personally see many potential targets for attack by INSM, 5) this case has just started- the trial in November is only the beginning- there will certainly be an appeal- I still believe INSM will prevail in November- I just don't think TRCA can defend the broadness of the patent- INSM will be able to pick and choose and put holes in TRCA defense.

Let's just hope the sales numbers look good and a breakthrough data for MMD od AIDS lipodystrophy data will be presented in the near future.

Good Luck to all longs and hope for better days to come. "

I finally had a chance to research this issue further. I believe the major concern for us all is whether the patent 414 that claims the method for IGF-1 production by TRCA is valid. The key question is "Is the method proposed in Patent 414 a novel method or novel invention for producing IGF-1" by DNA or TRCA. I am very relieved to say that the answer is a resounding "no". Following lies the evidence. I have searched the PUBMED and found over 10 prior publications which have used the identical method to produced IGF- that is utilizing the E.coli as the host and using recombinant DNA approach consisting of fusion protein and a plasmid vector. Following are six papers dating back as early as 1985: (I hope this makes your day). There is no way that TRCA can win the novel method patent- the key issue of this litigation.

1: Francis GL, Ross M, Ballard FJ, Milner SJ, Senn C, McNeil KA, Wallace JC,
King R, Wells JR.
Novel recombinant fusion protein analogues of insulin-like growth factor
(IGF)-I indicate the relative importance of IGF-binding protein and receptor
binding for enhanced biological potency.
J Mol Endocrinol. 1992 Jun;8(3):213-23.
PMID: 1378742 [PubMed - indexed for MEDLINE]

2: King R, Wells JR, Krieg P, Snoswell M, Brazier J, Bagley CJ, Wallace JC,
Ballard FJ, Ross M, Francis GL.
Production and characterization of recombinant insulin-like growth factor-I
(IGF-I) and potent analogues of IGF-I, with Gly or Arg substituted for Glu3,
following their expression in Escherichia coli as fusion proteins.
J Mol Endocrinol. 1992 Feb;8(1):29-41.
PMID: 1311930 [PubMed - indexed for MEDLINE]

3: Nilsson B, Forsberg G, Hartmanis M.
Expression and purification of recombinant insulin-like growth factors from
Escherichia coli.
Methods Enzymol. 1991;198:3-16. No abstract available.
PMID: 1857223 [PubMed - indexed for MEDLINE]

4: Hummel M, Herbst H, Stein H.
Gene synthesis, expression in Escherichia coli and purification of
immunoreactive human insulin-like growth factors I and II. Application of a
modified HPLC separation technique for hydrophobic proteins.
Eur J Biochem. 1989 Apr 1;180(3):555-61.
PMID: 2469575 [PubMed - indexed for MEDLINE]

5: Wong EY, Seetharam R, Kotts CE, Heeren RA, Klein BK, Braford SR, Mathis KJ,
Bishop BF, Siegel NR, Smith CE, et al.
Expression of secreted insulin-like growth factor-1 in Escherichia coli.
Gene. 1988 Sep 7;68(2):193-203.
PMID: 3065142 [PubMed - indexed for MEDLINE]

6: Moks T, Abrahmsen L, Holmgren E, Bilich M, Olsson A, Uhlen M, Pohl G, Sterky
C, Hultberg H, Josephson S, et al.
Expression of human insulin-like growth factor I in bacteria: use of optimized
gene fusion vectors to facilitate protein purification.
Biochemistry. 1987 Aug 25;26(17):5239-44.
PMID: 3676250 [PubMed - indexed for MEDLINE]

When I read the decision what I was struck with was the broadness and non-specificness of the patents. As this initial decision is just setting the ground rules, there are lot more twists and turns to come.

My initial impression was TRCA fared much better than INSM- which probably was the case. It appeared from the transcript that TRCA was better prepared than INSM. After having thought about this for most of the day (after being upset and down most of the day), I really feel that this initial decision was a non-event. The real trial is yet to come- nothing was settled here.

In reading the decision, it is quite apparent that the patents were broad as possible and I (not being a lawyer but a physician scientist) can already see many potential weaknesses with the position taken by TRCA. First, the patent for the production of IGF is as broad as can be- including usage of all prokaryotic host (not just E. coli). There are number of very broad application of the process that are mentioned throughout. There were some areas that the judge also had raised some issues for TRCA. The brief summary by Unterberg previously posted also provides some insight. SOme things that bother me about TRCA patent position are 1) the broadness of the patents in regards to production -they cover all prokaryotes (all bacterial hosts), 2) the broadness or nonspecific definition of IGF, fusion protein and etc..., 3) the method they described for IGF production is not unique or novel but a simple standard molecular methodology that is published in any first year college molecular biology textbook- it completely baffles me how this can be considered a patent but what do I know, 4) judge in this case just laid down the groundrules but did not entertain arguments as to why the patents are invalid- I personally see many potential targets for attack by INSM, 5) this case has just started- the trial in November is only the beginning- there will certainly be an appeal- I still believe INSM will prevail in November- I just don't think TRCA can defend the broadness of the patent- INSM will be able to pick and choose and put holes in TRCA defense.

Let's just hope the sales numbers look good and a breakthrough data for MMD od AIDS lipodystrophy data will be presented in the near future.

Good Luck to all longs and hope for better days to come.

Question for the board.

I read through the decision that was posted earlier. 414 patent specifically stated DNA vector in a "prokaryotic host cell" which implies all bacterial cells. This would be a problem even if Dow were able to produce IGF theorugh Pseudomonas -since Pseudomaonas is a prookaryotic cell. Any opinions.

Coninstarz,

You have done an amazing job with IHUB. I want to thank you fo all your hard work continually providing up-to-date info and balanced view to other INSM investors.

Your info regarding S-3 filing is very useful and keeps things in perspective.

I agree with everyone that at 2.3 PPS (market cap of about 100-150 mil), INSM is a bargain. The greatest risk to company, FDA and orphan drug approval, has passed and thus INSM is in the best shape that it could have hoped for- yet the comany has not been rewarded with a higher PPS. As noted by others, new risks and new challenges remain- however, these challenges and risks are minor compared to the initial risk of FDA approval- lack of approval would have been devastating to the company. Iplex approval was crucial in that it not only proves that Iplex is not only clinically useful (and superior to increlex), but allows INSM to generate revenue to support other more promising clinical trials. If Iplex indeed achieves sales of 100-200 million dollar per year, this should be sufficient to allow INSM to proceed with clinical trials without worrying about new funding.

I think the biggest potential positives for the near future are 1) announcement of pricing of Iplex (I personally believe, it will be priced over 22,000 per year- my guess is between 23-25K), 2) announcement of distribution and marketing infoplans ( possibly with new partners) and 3) info as to when Iplex will hit the market (it would be very important for Iplex to hit the market in 2nd quarter- if not this will be very negative). In the meantime there will be great deal of up and down movement of this stock with recent infusion of day traders.

IMO, the biggest near-term risk is the market penetration of the drug. The S-3 filing has addressed other important concern regarding financing. Although it has been perceived by many that S-3 filing is a negative- acquiring more fund for an emerging biotech comapny is never a bad thing IMO.

Good luck to all INSM investors.